RU2819613C1 - Bispecific antibodies to ccl2 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are: a bispecific anti-CCL2 antibody comprising first and second different antigen-binding sites binding to two different epitopes on human CCL2, and a method for producing same, recovered nucleic acid coding an antibody, a host cell, pharmaceutical compositions.

EFFECT: inventions are used as medicinal agents for treating cancer, inflammatory and autoimmune diseases.

18 cl, 33 dwg, 6 tbl, 4 ex

Description

The present invention relates to bispecific antibodies to CCL2 that bind to two different epitopes on human CCL2, their pharmaceutical compositions, their production and use as drugs for the treatment of cancer, inflammatory, autoimmune and ophthalmic diseases.

State of the art

The CCL2/CCR2 axis is a major mediator of immature myeloid cell recruitment to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), creating a chemoattractant gradient. Once reaching the tumor, myeloid suppressor cells (MSCs) become part of the protumorigenic environment by secreting/upregulating anti-inflammatory cytokines/receptors, which in turn inhibit the initiation of antitumor T cell responses. Thus, MSCs may reduce or even impair the effectiveness of T cell activating therapy (Meyer et al, 2014). Therefore, specifically inhibiting the recruitment of these immature myeloid cells would enhance the efficacy of checkpoint inhibitors, T-cell bispecific antibodies (TCBs), or other cancer immunotherapy (CI) options. In addition, CCL2 has also been associated with promotion of angiogenesis, metastasis, and tumor growth, suggesting that neutralization of CCL2 may be part of multiple lines of antitumor intervention.

Targeting CCL2 rather than its receptor will specifically inhibit unwanted CCL2-mediated effects without affecting those whose signaling may be through the same receptor (CCR2), but different ligands (e.g. CCL7, CCL8, CCL13) that are involved in recruiting populations of other immune cells such as Th1 and NK cells.

Clinically, CCL2 has been the preferred antibody target in several studies aimed at neutralizing its elevated levels caused by various inflammatory diseases, such as rheumatoid arthritis (Haringman et al, Arthritis Rheum. 2006 Aug;54(8):2387-92), idiopathic fibrosis lungs (Raghu et al, Eur Respir J. 2015 Dec;46(6): 1740-50), diabetic nephropathy (Menne et al, Nephrol Dial Transplant (2017) 32: 307-315) and cancer (Sandhu et al, Cancer Chemother Pharmacol 2013 Apr;71(4): 1041-50). However, its high rate of synthesis together with the high observed in vivo dissociation constants (KD) of the antibody antigen have been recognized as the main reasons preventing the suppression of free CCL2 by traditional antibodies at clinically acceptable doses (Fetterly et al, J Clin Pharmacol. 2013 Oct;53(10): 1020-7).

Neutralization of CCL2 appears to be more obviously relevant in patients with elevated serum levels of CCL2, which have been observed in several types of cancer, such as breast cancer (BC), ovarian cancer (OC), colorectal cancer (CR), pancreatic cancer, and prostate gland. However, even for patients with these indications who do not demonstrate similar serology but whose tumors are heavily infiltrated by immune cells of the myeloid lineage, this new therapy will be very beneficial due to the many roles that CCL2 plays in the tumor context, as mentioned above.

Igawa et al, Immunological Reviews 270 (2016) 132-151 describe sweep technology in which an engineered antibody carries a pH-dependent CDR (relating to the dissociation of antibody and antigen under the acidic conditions of endosomes, resulting in degradation of the antigen) and an engineered Fc- fragment with optimized isoelectric point (pI) and increased binding to FcgammaRIIb (which promotes cellular uptake of immune complexes) and has moderate affinity for the neonatal Fc receptor to maintain an acceptable pharmacokinetic profile.

The essence of the invention

The present invention relates to bispecific antibodies to CCL2 that bind to two different epitopes on human CCL2, their pharmaceutical compositions, their production and use as drugs for the treatment of cancer, inflammatory, autoimmune and ophthalmic diseases.

In one embodiment, the invention provides a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second distinct antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein the bispecific antibody comprises Fc domain of human IgG isotype.

In one embodiment, the invention provides a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second distinct antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

A) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

B) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

B) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; or

D) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

E) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (6) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46; or

E) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 51; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

G) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

And) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30;

or

K) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 3; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30.

In one embodiment, the bispecific antibody comprises an Fc domain of a human IgG isotype.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG isotype heavy chain constant domain.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain.

In one embodiment, the in vivo clearance rate (ml/day/kg) of human CCL2 following administration of a bispecific antibody comprising a wild-type human IgG1 isotype heavy chain constant domain (or Fc domain thereof) is at least twofold (in in one embodiment is at least 5 times greater, in one embodiment is at least 10 times greater, in one embodiment is at least 20 times greater) the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody containing the constant domain of the heavy chain of the human IgG1 isotype (or its Fc domain) with Fc-gamma receptor silencing, containing mutations L234A, L235A, P329G (EU numbering according to Kabat), when a preformed immune complex consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg human CCL2 were administered in a single dose of 10 ml/kg to FcRn transgenic mice.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein

i) said first antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35; and a VL domain containing the amino acid sequence of SEQ ID NO: 40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46.

In one embodiment, the in vivo clearance rate of human CCL2 (ml/day/kg) following administration of a bispecific antibody comprising the wild-type human IgG1 isotype heavy chain constant domain (or Fc domain thereof) is at least 15-fold greater than in particular, exceeds at least 20 times the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody containing the heavy chain constant domain of the human IgG1 isotype (or its Fc domain) with Fc gamma receptor silencing, containing mutations L234A, L235A, P329G (EU numbering according to Kabat), when a preformed immune complex consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg human CCL2 was administered in a single dose of 10 ml/kg to FcRn-transgenic mice.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) a CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ is V, I or H, X ² is P or H and X ³ is H or G, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; And

a VL domain containing (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) a CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ is V, I or H, X ² is P or H and X ³ is H or G, (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59, (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF from SEQ ID NO: 63, (e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG from SEQ ID NO: 64, (f) FR-H3 containing the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR from SEQ ID NO: 65, and (g) FR-H4 containing the amino acid sequence WGQGTLVTVSS from SEQ ID NO: 66; And

a VL domain containing(s) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (j) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (k) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62, (l) FR-L1 containing the amino acid sequence EIVLTQSPATLSLSPGERATLSC from SEQ ID NO: 67, (n) FR-L2 containing the amino acid sequence WYQQKPGQAPRLLIY from SEQ ID NO: 68, (p) FR-L3 containing the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC from SEQ ID NO: 69, and (p) FR-L4 containing amino acid sequence GQGTKVEIK from SEQ ID NO: 70; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D or E, (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E, (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS from SEQ ID NO: 82, ( e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG from SEQ ID NO: 83, (f) FR-H3 containing the amino acid sequence RVTITADTSSTAYMELSSLRSEDTAVYYCAR from SEQ ID NO: 84, and (g) FR-H4 containing the amino acid sequence WGQGTTVTVSS from SEQ ID NO: 85; And

a VL domain containing(s) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (j) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, (k) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R, (m) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC from SEQ ID NO: 86, (n) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIH from SEQ ID NO: 87, (p) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC from SEQ ID NO: 88, and (p) FR-L4 containing amino acid sequence FGGGTKVEIK from SEQ ID NO: 89.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

A) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO:90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

D) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

E) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO:90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

E) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

G) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

And) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

K) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

K) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

M) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

H) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

P) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

P) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

C) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

T) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

A) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, X ² represents P and X represents H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F and X ² is R, (e) CDR-L2 containing the amino acid the sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W;

or

B) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X represents I, (b) CDR-H2 containing the amino acid sequence

GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ is V, X ² is P and X ³ is H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F and X ² is R, (e) CDR-L2 containing the amino acid the sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W; or

B) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

D) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

E) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

E) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

G) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

i) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

K) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X is R or L, (e) CDR-L2 , containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R;

or

K) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

M) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

H) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R; or

P) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

P) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

C) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

T) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

In one embodiment, a bispecific antibody described herein

i) blocks the binding of CCL2 to its receptor CCR2 in vitro (reporter gene assay, IC ₅₀ =0.5 nM); and/or

ii) inhibits CCL2-mediated chemotaxis of myeloid cells in vitro (IC ₅₀ =1.5 nM); and/or

iii) is cross-reactive with cynomolgus and human CCL2.

In one embodiment, the bispecific antibody described herein is not cross-reactive with other CCL homologs, and in particular, it exhibits 100-fold less binding to other CCL homologs (such as, for example, CCL8) compared to binding to CCL2.

In one embodiment, a bispecific antibody described herein binds to the first and second epitopes on human CCL2 in an ion-dependent manner.

In one embodiment, a bispecific antibody described herein binds to human CCL2 in a pH-dependent manner, and wherein both the first antigen binding site and the second antigen binding site bind to CCL2 with greater affinity at neutral pH than at acidic pH.

In one embodiment, a bispecific antibody described herein binds to human CCL2 with 10 times greater affinity at pH 7.4 than at pH 5.8.

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and/or ADZOK (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L235W, G236N, H268D, Q295L, K326T and/or AZZOK (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pi to increase antigen uptake); And

ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iii) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein contains an IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering) Q311R and P343R (suitable for increasing the pI to enhance antigen uptake).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L234Y, P238D, T250V, V264I, T307P and/or A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) M428L, N434A and Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises two IgG1 heavy chain constant domains (or Fc domain thereof) containing (independent of or in addition to the mutations described above) the following mutations (Kabat EU numbering)

i) S354C and T366W in one of the heavy chain constant domains,

ii) Y349C, T366S, L368A, Y407V in another of the heavy chain constant domains.

In one embodiment, the invention provides an (isolated) (monospecific) antibody that (specifically) binds to human CCL2, wherein the antibody comprises

A) a VH domain containing (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, wherein X ¹ is V, I or H, X ² is P or H and X ³ is H or G, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; or

B) a VH domain containing (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X represents D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E; And

a VL domain containing (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

In one embodiment, the invention provides an (isolated) (monospecific) antibody that (specifically) binds to human CCL2, wherein the antibody comprises

A) a VH domain containing the amino acid sequence of SEQ ID NO: 71; And

VL domain containing the amino acid sequence SEQ ID NO: 75;

or

B) VH domain containing the amino acid sequence SEQ ID NO: 72;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75;

or

B) VH domain containing the amino acid sequence SEQ ID NO: 73;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75;

or

D) VH domain containing the amino acid sequence SEQ ID NO: 74;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75;

or

E) VH domain containing the amino acid sequence of SEQ ID N0:90;

and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

E) a VH domain containing the amino acid sequence of SEQ ID NO: 91;

and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

G) VH domain containing the amino acid sequence SEQ ID NO: 92;

and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

I) VH domain containing the amino acid sequence of SEQ ID N0:90;

and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

In one embodiment, the invention provides an isolated nucleic acid encoding a (mono- or bispecific) antibody in accordance with any of the previous embodiments.

In one embodiment of the invention, a host cell containing such a nucleic acid is provided.

In one embodiment of the invention, a method for producing an antibody is provided, comprising culturing such a host cell to produce the antibody.

In one embodiment of the invention, such a method further includes the step of isolating the antibody from the host cell.

In one embodiment, the invention provides a pharmaceutical composition comprising a bispecific antibody described herein and a pharmaceutically acceptable carrier.

In one embodiment, the invention provides a bispecific antibody described herein for use as a drug.

In one embodiment, the invention provides the use of a bispecific antibody described herein in the manufacture of a drug.

In one embodiment, such a drug is for the treatment of cancer.

In one embodiment, such a drug is for the treatment of an inflammatory or autoimmune disease.

In one embodiment, the invention provides a bispecific antibody described herein for use in the treatment of cancer.

In one embodiment, the invention provides a bispecific antibody described herein for use in the treatment of an inflammatory or autoimmune disease.

In one embodiment, the invention provides a method of treating an individual having cancer, comprising administering to the individual an effective amount of an antibody described herein.

In one embodiment, the invention provides a method of treating an individual having an inflammatory or autoimmune disease, comprising administering to the individual an effective amount of an antibody described herein.

Brief description of graphic materials

Fig. 1: Surface plasmon resonance (Biacore®) sensorgrams showing binding of monospecific CCL2 antibodies (CNT0888 (=CNTO), 1A5, 1G9 and humanized 11K2 (=11k2) to recombinant CCL2 and CCL2 homologs.

Fig. 2a: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody CNT0888-SG1 (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody CNTO888-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 2b: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific antibody to CCL2 11K2-SG1 (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 2c: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody ABN912-SG1 (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody ABN912-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 2d: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific antibody to CCL2 1A4-SG1 (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1A4-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 2e: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific antibody to CCL2 1A5-SG1 (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1A5-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 2f: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1G9-SGI (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1G9-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 2g: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific antibody to CCL2 2F6-SG1 (IgG1 wild type), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 2F6-SG105 (Fc receptor binding silencing IgG1), FcRn transgenic mice.

Fig. 3: Illustrates the time course of mouse total serum CCL2 concentration (Figure 3a) and antibody-time profile (Figure 3b) after IV injection a) solid line: 20 mg/kg monospecific anti-CCL2 antibody 11K2-SG1 (wild-type IgG1 ), and b) dotted line: 20 mg/kg monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor binding silencing IgG1) in mice.

Fig. 4a: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 11K2//1G9- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 11K2//1G9-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4b: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 CNTO888//11K2- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody CNTO888//11K2-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4c: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 CNTO888//1G9- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 11K2//1G9-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4d: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 CNTO888//1A5- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody CNTO888//1A5-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4e: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 1A5//1G9- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 1A5//1G9-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4f: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 11K2//2F6- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 11K2//2F6-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4g: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 ABN912//11K2- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 ABN912//11K2-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4i: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 1A4//2F6- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 1A4//2F6-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 4j: Serum concentration of hCCL2 over time after IV injection of a preformed immune complex consisting of a) solid line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific antibody to CCL2 1A5//2F6- WT IgG1 (wild type IgG1 with intact Fc receptor binding), or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody 1A5//2F6-PGLALA (silenced IgG1 Fc receptor binding), Balb/c mice.

Fig. 5a: Biacore® sensorgrams showing the binding profile to monomeric CCL2 at pH 7.4 (black line) and pH 5.8 (gray line) of four modified 11K2 and four CNT0888 variants, as well as 16 bispecific antibodies to CCL2, CKLO01 to CKLO16 , producing a corresponding combination of antigen-binding fragments of four modified 11K2 and four CNTO888 variants.

Fig. 5b: Biacore® sensorgrams showing the binding profile to monomeric CCL2 of four modified 11K2 and four variants of CNTO888, as well as 16 bispecific antibodies to CCL2, CKLO01 to CKLO16, producing the corresponding combination of antigen binding fragments of four modified 11K2 and four variants of CNTO888. An additional dissociation phase at pH 5.8 was integrated into the BIACORE® assay immediately after the dissociation phase at pH 7.4.

Figure 6: Biacore® sensorgrams showing the binding profile of CCL2 bispecific antibodies CKLO01, CKLO02, CKLO03 and CKLO04 to monomeric CCL8 at pH 7.4 (black line) and pH 5.8 (gray line).

Fig. 7a: Serum concentration of hCCL2 over time after injection of a preformed immune complex consisting of hCCL2 and bispecific antibodies to CCL2 (parental CNTO//11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03 and CKLO04) into SCID mice.

Fig. 7b: Serum concentration of hCCL2 over time after injection of a preformed immune complex consisting of hCCL2 and CKLO03 (with wild-type IgG1 Fc) or CKLO03-SG1099 (CKLO03 with elevated pI Fc) into SCID mice.

Fig. 8: Chemotactic assay: Bispecific antibodies to CCL2 with identical CDR and VH/VL variable regions, namely wild-type CKLO2-IgG1 and CKLO2-SG1095, but with different Fc fragments, can inhibit THP-1 cell migration with identical potency (IC ₅₀ =0.2 μg/ml; Fig. 8, left panel).

Similarly, CCL2-0048, the parent unmodified CNTO888/1 Ik2k2 IgG1 bispecific antibody from CKLO2, which is not pH dependent, also exhibits an IC ₅₀ of 0.2 μg/mL, since pH dependence is critical for antigen sweeping, a phenomenon that is not observed in this analysis.

The corresponding monospecific antibodies CNTO888 IgG1 and humanized 11k2 IgG1 showed IC ₅₀ values of 0.3 and 0.7 μg/ml, respectively, while the isotype control huIgG1 showed no inhibition (Fig. 8, right panel).

Fig. 9: In vivo antitumor activity in a genetically modified mouse model. Treatment of mouse tumor model with Mab CKLO2-IgG1 (IgG1 with wild-type Fc) and CKLO2-SG1099 ((=CKLO2 with increased pI). Tumor volume (left), tumor mass (middle) and M-MSC infiltrate (right) at the end studies, (carrier is shown in black, wild type IgG1 CKLO2 is gray, and CKLO2 with Fc with increased pI (CKLO2-SG1099) is white bars/dashed line)

Fig. 10: Serum levels of total (left) and free (right) CCL2 during an in vivo study of antitumor activity (see efficacy in Fig. 9) when treated with bispecific antibodies to CCL2 (vehicle depicted in black, wild-type IgG1 CKLO2 in gray, and Fc with increased pI (CKLO2-SG1099) white bars/dashed line).

Fig. 11: Proof-of-concept study of the efficiency of CCL2 sweeping in cynomolgus monkeys. Total antibody concentration-time profiles in cynomolgus monkey serum; left panel: mean concentration-time profiles of four antibodies presented over seven days; group 1: monospecific CNTO888-SG1 (= wild-type IgG1) antibody to CCL2 (n=3 animals) as a control for maximum accumulation of total CCL2; group 2: anti-CCL2 antibody with two paratopes CKLO2-SG1 (wild-type IgG1) with pH-dependent target binding, but without Fc modifications (n=3); group 3: anti-CCL2 antibody with two paratopes CKLO2-SG1100 with pH-dependent target binding and Fc-pI and additional modifications (n=4) and group 4: anti-CCL2 antibody with two paratopes CKLO2-SG1095 with pH-dependent target binding, with Fc-pI modifications and increasing affinity for FcγRIIb and additional modifications (n=4); right panel: individual concentration time profile of individual 4 (group 2) presented during the PK study (70 days).

Fig. 12: Proof-of-concept study of the efficiency of CCL2 sweeping in cynomolgus monkeys. Total CCL2 concentration time profiles in cynomolgus monkey serum; left panel: mean total CCL2 concentration-time profiles of four antibodies presented over seven days; right panel: individual total CCL2 concentration-time profile of individual 4 (group 2) presented during the PK study (70 days).

Fig. 13: Free CCL2 concentration time profiles in cynomolgus monkey serum; left panel: mean free CCL2 concentration-time profiles of four antibodies presented over seven days; right panel: individual total CCL2 concentration profile - time of individual 4 (group 2) presented during the PK study (70 days); average profiles were calculated using a value of 0.01 ng/mL (lower limit of quantitation) for samples that were below the limit of detection.

Fig. 14: PK/PD study of the efficiency of CCL2 sweeping in cynomolgus monkeys. Concentration profiles of total CKL02-SG1095 time in the serum of cynomolgus monkeys (treatment with CKL02-SG1095 at different concentrations (groups 1-3)); left panel: mean concentration-time profiles (n=4) for three dose levels are presented over seven days; right panel: individual concentration-time profiles of two ADA-negative individual animals (25 mg/kg dose group) are presented over the course of the study (98 days).

Fig. 15: PK/PD study of the efficiency of CCL2 sweeping in cynomolgus monkeys. Concentration profiles of total CCL2 time in the serum of cynomolgus monkeys treated with CKL02-SG1095 at different concentrations (groups 1-3) and compared with treatment with CNT0888-SG1 (group 4); left panel: mean total CCL2 concentration time profiles (error bars indicate SD) of the four study groups are presented over seven days; right panel: individual total CCL2 concentration time profiles of ADA-negative animals from groups 3 (n=2, error bars indicate range) and 4 (n=3, error bars indicate SD) are presented during the PK study (98 days).

Fig. 16: PK/PD study of the efficiency of CCL2 sweeping in cynomolgus monkeys. Free CCL2 concentration time profiles in cynomolgus monkey serum; left panel: mean free CCL2 concentration time profiles (error bars indicate SD) of the four study groups are presented over seven days (CKL02-SG1095 treatment at different concentrations (groups 1-3) and compared with CNT0888-SG1 treatment (group 4)); right panel: mean free CCL2 concentration time profiles of ADA-negative animals from groups 3 (n=2, error bars indicate range) and 4 (n=3, error bars indicate SD) are presented during the PK study (70 days); average profiles were calculated using a value of 0.01 ng/mL (lower limit of quantitation) for samples that were below the limit of detection.

Detailed description of the invention

The present invention relates to bispecific antibodies to CCL2 that bind to two different epitopes on human CCL2, their pharmaceutical compositions, their production and use as drugs for the treatment of cancer, inflammatory, autoimmune and ophthalmic diseases. Thus, the antibody contains a first antigen binding site that (specifically) binds to a first epitope on human CC2, and a second distinct antigen binding site that (specifically) binds to a distinct second epitope.

The present invention includes bispecific antibodies comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

i) said first antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35; and a VL domain containing the amino acid sequence of SEQ ID NO:40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46.

In one embodiment, the in vivo clearance rate of human CCL2 (ml/day/kg) following administration of a bispecific antibody comprising the wild-type human IgG1 isotype heavy chain constant domain (or Fc domain thereof) is at least 15-fold greater than in particular, exceeds at least 20 times the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody containing the heavy chain constant domain of the human IgG1 isotype (or its Fc domain) with Fc gamma receptor silencing, containing mutations L234A, L235A, P329G (EU numbering according to Kabat), when a preformed immune complex consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg human CCL2 was administered in a single dose of 10 ml/kg to FcRn-transgenic mice.

The term "epitope" includes any polypeptide determinant capable of specifically binding to an antibody. In some embodiments, the epitope determinant contains reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in some embodiments may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody. The present invention provides bispecific anti-CCL2 antibodies that bind to two different epitopes on human CCL2. The terms first and second epitope refer to two different epitopes on human CCL2. Thus, the second epitope is different from the first epitope. Whether an antibody binds to the same epitope as or competes with the reference antigen binding site for CCL2 can be readily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as the CCL2 reference antigen binding site of the invention, the reference antibody is allowed to bind to the CCL2 domain under saturated conditions. Next, the ability of the test antibody to bind to human CCL2 is assessed. If the test antibody is able to bind to human CCL2 after saturating binding to the CCL2 reference antigen binding site, it can be concluded that the test antibody binds to a different epitope than the CCL2 reference antigen binding site. Alternatively, if the test antibody is unable to bind to human CCL2 after saturating binding to a reference anti-CCL2 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-CCL2 antibody of the invention. Additional routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm that the observed lack of binding is indeed due to binding to the same epitope as the reference antibody, or that steric blocking (or other phenomenon). Experiments of this nature can be performed using ELISA, RIA, surface plasma resonance (eg, Biacore), flow cytometry, or any other qualitative or quantitative antibody binding assay available in the art. In accordance with certain embodiments of the present invention, two antibodies bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502).

Alternatively, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are considered to have “overlapping epitopes” if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

To determine whether an antibody competes for binding to a reference antibody to CCL2, the above-described binding methodology is carried out in two variations: In the first embodiment, the reference antibody is allowed to bind to CCL2 under saturating conditions and then assessed for binding of the test antibody to human CCL2. In the second embodiment, it is possible to bind the test antibody to the CCL2 molecule under saturating conditions, followed by assessment of the binding of the reference antibody to human CCL2. If in both cases only the first (saturating) antibody is able to bind to the CCL2 molecule, it is concluded that the test antibody and the reference antibody compete for binding to CCL2. As one skilled in the art will appreciate, an antibody that competes for binding to a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

As used herein, the term “CCL2,” “human CCL2,” also referred to as “MCP-1,” refers to the 76-amino acid sequence identified in NCBI entry No. NP_002973 and known by various names such as CCL2, MCP-1 (monocyte chemotactic protein 1), SMC-CF (smooth muscle cell chemotactic factor), LDCF (lymphocyte chemotactic factor), GDCF (glioma monocyte chemotactic factor), TDCF (tumor chemotactic factor), HCl1 (human cytokine 11), MCAF (monocyte chemotactic and activating factor) . The gene symbol is SCYA2, the JE gene on human chromosome 17, and the new designation is CCL2 (Ziotnik, Yoshie 2000. Immunity 12: 121-127). JE is the mouse homologue of human MCP-1/CCL2.

Handel and others (Biochemistry. 1996; 35:6569-6584) determined the structure of the CCL2 dimer in solution. These studies revealed that the secondary structure of CCL2 consists of four β-sheets. Additionally, residues responsible for the dimerization surfaces of CCL2 were described by Zhang and Rollins (Mol Cell Biol. 1995; 15:4851-4855). The protein complex has an elongated shape with two monomers oriented to form a large pocket. The structures of monomeric and dimeric CCL2 in two crystal forms, the so-called I and P forms, have also been determined (Lubkowski et al., Nat Struct Biol. 1997; 4:64-69). Paolini et al, (J Immunol. 1994 Sep 15; 153(6):2704-17) describe that MCP1/CCL2 exists as a monomer at physiologically relevant concentrations:

Through river analysis. protein CCL2 (purchased from Peprotech) using size exclusion HPLC, sedimentation equilibrium ultracentrifugation and chemical cross-linking, they were able to show that the mass fraction of monomeric and dimeric forms of MCP-1 is concentration dependent in vitro. Finally, Seo et al (J Am Chem Soc. 2013 Mar 20; 135(11):4325-32) were able to demonstrate the presence of administered CCL2 in both monomeric and dimeric forms under physiological conditions using ion mobility mass spectrometry.

Thus, “wild-type CCL-2” (CCL2 wt) can exist as a monomer, but in fact can also form dimers at physiological concentrations. This equilibrium between monomers and dimers is certainly different and must be carefully considered for all described in vitro experiments in which different concentrations may be used. To avoid uncertainty, we have generated CCL2 variants with point mutations: The “P8A” variant of CCL2 carries a mutation in the dimerization interface that results in an inability to form a dimer, resulting in a certain pure CCL2 monomer. In contrast, the “T10C” variant of CCL2 results in a fixed CCL2 dimer (J Am Chem Soc. 2013 Mar 20; 135(11):4325-32).

The CCL2/CCR2 axis is a major mediator of immature myeloid cell recruitment to tumors. CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM), creating a chemoattractant gradient. Once reaching the tumor, myeloid suppressor cells (MSCs) become part of the protumorigenic environment by secreting/upregulating anti-inflammatory cytokines/receptors, which in turn inhibit the initiation of antitumor T cell responses. Thus, MSCs may reduce or even impair the effectiveness of T cell activating therapy (Meyer et al, 2014). Therefore, specifically inhibiting the recruitment of these immature myeloid cells will enhance the effectiveness of checkpoint inhibitors, T-cell bispecific antibodies, and cancer immunotherapy options. In addition, CCL2 has also been associated with promotion of angiogenesis, metastasis, and tumor growth, suggesting that neutralization of CCL2 may be part of multiple lines of antitumor intervention.

Targeting CCL2 rather than its receptor will specifically inhibit unwanted CCL2-mediated effects without affecting those whose signaling may be through the same receptor (CCR2), but different ligands (e.g. CCL7, CCL8, CCL13) that are involved in recruiting populations of other immune cells such as Th1 and NK cells.

Clinically, CCL2 has been the preferred antibody target in several studies aimed at neutralizing its elevated levels caused by various inflammatory diseases, such as rheumatoid arthritis (Haringman et al, 2006), idiopathic pulmonary fibrosis (Raghu et al, 2015), diabetic nephropathy (Menne et al, 2016) and cancer (Sandhu et al, 2013). However, its high rate of synthesis together with the high observed in vivo dissociation constants (KD) of the antibody antigen have been recognized as the main reasons preventing the suppression of free CCL2 by traditional antibodies at clinically acceptable doses (Fetterly et al, 2013).

Neutralization of CCL2 appears to be more obviously relevant in patients with elevated serum levels of CCL2, which have been observed in several types of cancer, such as breast cancer (BC), ovarian cancer (OC), colorectal cancer (CR), pancreatic cancer, and prostate gland. However, even for patients with these indications who do not demonstrate similar serology but whose tumors are heavily infiltrated by immune cells of the myeloid lineage, this new therapy will be very beneficial due to the many roles that CCL2 plays in the tumor context, as mentioned above.

As used herein, an antibody “binding to human CCL2”, “specifically binding to human CCL2”, “which binds to human CCL2”, or “anti-CCL2 antibody” refers to an antibody that specifically binds to a human CCL2 antigen with a binding affinity of k _d 5.0 x 10 ^-8 mol/L or lower, in one embodiment with a k _d value of 1.0 x 10 ^-9 mol/L or lower, in one embodiment with a k _d value of 5.0 x 10 ^-8 mol/l to 1.0 x 10 ^-13 mol/l.

Binding affinity is determined using a standard binding assay, such as the surface plasma resonance method (BIAcore®, GE-Healthcare Uppsala, Sweden), for example using constructs containing the extracellular domain of CCL2 (eg, in its naturally occurring 3-dimensional structure). In one embodiment, binding affinity is determined using a standard binding assay using soluble CCL2.

Antibody specificity refers to the selective recognition by an antibody of a specific epitope of an antigen. Natural antibodies, for example, are monospecific.

As used herein, the term “monospecific” antibody means an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen.

As used herein, the term “bispecific antibody that binds to CCL2 (human)”, “dual paratope antibody that binds to CCL2 (human)”, “bispecific antibody to CCL2”, “dual paratope antibody to CCL2” means, that the antibody is capable of specifically binding to at least two different epitopes on (human) CCL2. Typically, such a bispecific antibody contains two different antigen binding sites (two different paratopes), each of which is specific for a different epitope on CCL2 (human). In certain embodiments, the bispecific antibody is capable of binding two different and non-overlapping epitopes on CCL2, meaning that two different antigen binding sites do not compete for binding to CCL2.

For the purposes of this document, a “human acceptor framework” is a framework containing the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human acceptor framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the human VL acceptor framework is identical in sequence to a human immunoglobulin VL framework sequence or a human consensus framework sequence.

The term “antibody” is used herein in its broadest sense to include various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the required antigen binding activity.

"Antibody fragment" refers to a molecule, other than the intact antibody, that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

As used herein, the term “valency” refers to the presence of a certain number of antigen binding sites in an antibody. Therefore, the term “monovalent antigen binding” refers to the presence of one (and no more than one) antigen-specific antigen-binding site in an antibody.

The term "antigen binding site" refers to a site or region, i.e. one or more amino acid residues of an antibody that mediate interaction with the antigen. For example, the antigen binding site of an antibody contains amino acid residues from complementarity determining regions (CDRs). In one embodiment, the antigen binding site of the antibody comprises amino acid residues from VH and VL. The native immunoglobulin molecule typically has two antigen-binding sites; a Fab molecule typically has one antigen-binding site. "Antigen-binding fragment" refers to a polypeptide molecule containing an antigen-binding site that specifically binds to an antigenic determinant. Antigen binding fragments include antibodies and fragments thereof as further defined herein. Specific antigen binding fragments include an antibody antigen binding domain comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, antigen binding fragments may comprise antibody constant regions, as further defined herein and known in the art. Useful heavy chain constant regions are any of five isotypes: α, δ, ε, γ, or μ. Applicable light chain constant regions belong to either of two isotypes: κ and λ.

As used herein, the term “antigenic determinant” or “antigen” refers to a site on a polypeptide macromolecule to which an antigen binding moiety/site binds to form an antigen binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other disease-affected cells, on the surface of immune cells, free in blood serum and/or in the extracellular matrix (ECM).

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from another source or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain contains. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Preferably, the bispecific antibodies of the invention are of the human IgG isotype, more preferably the human IgG1 isotype. As used herein, the terms IgG isotype and IgG1 isotype refer to human IgG isotype and human IgG1 isotype. Typically, different IgG isotypes exist in the form of slightly different allelic variation-based allotypes among IgG subclasses (see Vidarsson et al.; Front Immunol 5 (2014) Article 520, 1-17). An "effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective at the required dosages and for the required periods of time to achieve the desired therapeutic or prophylactic result.

As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the IgG heavy chain Fc region can vary slightly, the human IgG heavy chain Fc region, by definition, generally extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational elimination of one or more, in particular one or two; amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell through expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain a full-length heavy chain or may contain a cleaved version of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This can occur when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU numbering). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) Fc regions may or may not be present. Heavy chain amino acid sequences, including Fc domains (or an Fc domain subunit as defined herein), are referred to herein without the C-terminal glycine-lysine dipeptide unless otherwise indicated. In one embodiment of the invention, the heavy chain containing the Fc domain subunit as defined herein contained in an antibody or bispecific antibody according to the invention contains an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index according to Kabat). In one embodiment, the heavy chain containing the Fc domain subunit as defined herein contained in an antibody or bispecific antibody according to the invention contains an additional C-terminal glycine residue (G446, Kabat EU numbering). The compositions of the invention, such as the pharmaceutical compositions described herein, contain a population of antibodies or bispecific antibodies of the invention. The population of antibodies or bispecific antibodies may contain molecules having a full-length heavy chain and molecules having a split version of the heavy chain. The population of antibodies or bispecific antibodies may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved version of the heavy chain, with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antibodies have a cleaved heavy chain variant. In one embodiment, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain containing an Fc domain subunit as defined herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering in accordance with the EU index according to Kabat). In one embodiment, a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain containing an Fc domain subunit as defined herein with an additional C-terminal glycine residue (G446, numbered according to EU index according to Kabat). In one embodiment of the invention, the composition contains a population of antibodies or bispecific antibodies consisting of molecules containing a heavy chain containing an Fc domain subunit as defined herein; molecules containing a heavy chain containing an Fc domain subunit as defined herein with an additional C-terminal glycine residue (G446, Kabat EU numbering); and molecules containing a heavy chain containing an Fc domain subunit as defined herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the “EU numbering system,” also referred to as “Kabat EU index numbering” or “Kabat EU numbering,” described in Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a “subunit” of an Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e. a polypeptide containing the C-terminal constant regions of the immunoglobulin heavy chain, capable of stable self-association. For example, the Fc domain subunit of an IgG contains the IgG CH2 and IgG CH3 constant domains.

"Framework region" or "FR" refers to variable domain residues other than hypervariable region residues (HVR). The FR variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Accordingly, the CDR and FR sequences are usually found in the VH (or VL) in the following order: FR-H1(L1)-CDR-H1(L1)-FR-H2(L2)-CDR-H2(L2)-FR-H3( L3)-CDR-H3(L3)-FR-H4(L4).

As used herein, the terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably to refer to an antibody having a structure substantially similar to that of a native antibody or having heavy chains that contain an Fc region as defined herein. document.

A “human antibody” is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or obtained from a non-human source that uses human antibody repertoires or other sequences encoding human antibodies. This definition of a human antibody expressly excludes a humanized antibody containing non-human antigen-binding moieties.

A “human consensus framework region” is a framework region that represents the most frequently occurring amino acid residues in a set of human immunoglobulin VL or VH framework sequences. Typically, a set of human immunoglobulin VL or VH sequences is derived from a subset of variable domain sequences. In general, a subgroup of sequences is a subgroup according to Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, in the case of VL, the subgroup is kappa I subgroup according to Kabat et al., supra. In one embodiment, in the case of VH, the subgroup is subgroup III of Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody containing amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one and typically two variable domains, in which all or substantially all of the CDRs correspond to those of the non-human antibody and all or substantially all of the FRs correspond to those of the human antibody. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of an antibody, for example a non-human antibody, refers to an antibody that has been humanized.

As used herein, the term “complementarity determining regions” or “CDRs” refers to each of the regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”), and/or contain antigen-contacting residues (“antigenic contacts”). In general, antibodies contain six CDRs: three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Typical CDRs for this document include:

(a) hypervariable loops located in amino acid residues 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55 ( CDR-H2) and 96-101 (CDR-H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs located in amino acid residues 24 34 (CDR-L1), 50 56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2 ) and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigenic contacts located in amino acid residues 27 c-36 (CDR-L1), 46 55 (CDR-L2), 89 96 (CDR-L3), 30 35b (CDR-H1), 47 58 (CDR-H2 ) and 93-101 (CDR-H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); And

(d) combinations (a), (b) and/or (c), including amino acid residues CDR 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (vL3), 31-35 (CDR-H1), 50-63 (CDR-H2) and 95,102 (CDR-H3).

Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein in accordance with Kabat et al., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs and horses), primates (such as humans and non-human primates such as monkeys), rabbits and rodents (such as mice and rats) . In certain embodiments, the individual or subject is a human.

An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity by definition, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. In 848 (2007) 79-87.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal position that differs from its natural chromosomal position.

"Isolated nucleic acid encoding a mono- or bispecific antibody to CCL2" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including nucleic acid molecules in the same vector or separate vectors and nucleic acid molecules, present at one or more locations in the host cell.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies composing the population are identical and/or bind to the same epitope, with the exception of possible variant antibodies, for example those containing mutations that are naturally occurring or arise during production of the monoclonal antibody preparation, with such variants generally present in minor numbers quantities. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the designation “monoclonal” refers to the characterization of an antibody as being derived from an essentially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced using a variety of techniques, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and techniques using transgenic animals containing all or part of the human immunoglobulin loci , such methods and other exemplary methods for producing monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins weighing about 150,000 Daltons, consisting of two identical light chains and two identical heavy chains that are linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Likewise, in the N-terminal to C-terminal direction, each light chain contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a light chain constant domain (CL). An antibody light chain can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions typically included in commercial packaging of therapeutic products that provide information regarding the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

"Percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after sequence alignment and gapping, if necessary, to achieve maximum percentage identity sequences, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For the purposes of this document, percent (%) amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code has been filed with user documentation at the United States Copyright Office, Washington, DC 20559, where it is registered under United States Copyright Number TXU510087. The ALIGN-2 program is freely available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

When ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A with or compared to a given amino acid sequence B (which can alternatively be stated as a given amino acid sequence A that has or contains a certain % The identity of an amino acid sequence with or compared to a given amino acid sequence B) is calculated as follows:

100 times the X/Y ratio,

where X represents the number of amino acid residues judged by the ALIGN-2 sequence alignment program to be identical matches in the software alignment of A and B, and where Y represents the total number of amino acid residues in B. It should be understood that in the case where the length of the amino acid sequence A is not equal length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are derived as described in the previous paragraph with using the ALIGN-2 computer program.

The term "pharmaceutical composition" refers to a preparation that is in a form that provides the biological activity of the active ingredient it contains, and which does not contain additional components that are unacceptably toxic to the subject to whom the composition will be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.

In the context of this document, the term “treatment” (and its grammatical variants such as “treat” or “treating”) refers to a clinical intervention with the purpose of changing the natural history of the disease of the individual being treated, and can be carried out either preventively or during treatment. clinical pathology. Desired effects of treatment include, but are not limited to, prevention of the onset or reappearance of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, reduction in the intensity or temporary relief of the disease state, and remission or improvement forecast. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (CDR). (See, for example, Kindt, T.J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

As used herein, the term “vector” refers to a nucleic acid molecule capable of increasing the copy number of another nucleic acid to which it is linked. The term includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it is introduced. Some vectors are capable of directing the expression of the nucleic acids to which they are operably linked. Such vectors are referred to herein as “expression vectors.”

I. COMPOSITIONS AND METHODS

In one aspect, the invention is based in part on the discovery that the bispecific antibodies described herein use different antigen binding sites for CCL2 as the first and second antigen binding site/fragment. These anti-CCL2 antibodies bind to specific epitopes of CCL2 with high specificity and have the ability to specifically inhibit the binding of CCL2 to its receptor CCR2. They demonstrate improved immune complex formation compared to monospecific antibodies and improved clearance of CCL2 in vivo.

Bispecific antibodies to CCL2

Bispecific antibodies

The bispecific antibodies described herein are monoclonal antibodies that have binding specificity for at least two different epitopes on CCL2.

Techniques for creating multi- and bispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), and overhang design into the cavity" (see, for example, US Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be created by design using the effect of electrostatic interaction to create Fc heterodimeric antibody molecules (see, for example, WO 2009/089004); cross-linking two or more antibodies or fragments (see, for example, US Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); the use of leucine zippers for the production of bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011/034605); the use of common light chain technology to overcome the problem of light chain mispairing (see, for example, WO 98/50431); the use of diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single chain Fv dimers (sFv) (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

Engineered antibodies with three or more antigen binding sites, including, for example, "octopus antibodies" or DVD-Ig, are also included herein (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. The bispecific antibody or antigen-binding fragment thereof also includes a "dual-action FAb" or "DAF" containing an antigen-binding site that binds to CCL2 as well as another different antigen, or two different epitopes of CCL2 (see, for example, US 2008/0069820 and WO 2015/095539).

Multispecific antibodies can be provided in an asymmetric form with crossover domains in one or more binding arms with the same antigen specificity, i.e. by exchanging VH/VL domains (see eg WO 2009/080252 and WO 2015/150447), CH1/CL domains (see eg WO 2009/080253) or full Fab arms (see eg WO 2009/080251 , WO 2016/016299, also see Schaefer et al., PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20), also called CrossMab. Asymmetric binding arms can also be constructed by introducing charged or uncharged amino acid mutations into the contact surfaces of the domains to ensure proper Fab pairing. See for example WO 2016/172485.

Various additional molecular formats for multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

Preferred formats of bispecific antibodies.

In accordance with specific embodiments of the invention, a bispecific antibody described herein is provided with crossover domains in one or more binding arms with the same antigen specificity, i.e. by exchanging VH/VL domains (see eg WO 2009/080252 and WO 2015/150447), CH1/CL domains (see eg WO 2009/080253) or full Fab arms (see eg WO 2009/080251 , WO 2016/016299, also see Schaefer et al., PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20).

Charge modifications in such bispecific antibodies, especially those containing VH/VL domain exchange (see WO 2015/150447):

The bispecific antibodies of the invention may contain amino acid substitutions in the Fab molecules they contain, which are particularly effective in reducing the mispairing of light chains with mismatched heavy chains (Bence-Jones type by-products) that can occur when preparing bispecific antibodies based on Fabs with VH/VL exchange in one (or more in the case of molecules containing more than two antigen-binding Fab molecules) of their binding arms (also see PCT Publication No. WO 2015/150447, incorporated herein by reference in its entirety, particularly examples in her). The ratio of desired bispecific antibody versus unwanted by-products, particularly Bence-Jones-type by-products arising in bispecific antibodies with a VH/VL domain exchange in one of their binding arms, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as “charge modifications”).

Accordingly, in some embodiments, in which the first and second antigen binding fragments of the bispecific antibody are both Fab molecules, and in one of the antigen binding fragments (particularly, the second antigen binding fragment), the VL and VH variable domains of the Fab light chain and Fab heavy chain are replaced each other,

i) in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat), and in the CH1 constant domain of the first antigen binding fragment, the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbering in accordance with the EU index according to Kabat); or

ii) in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat), and in the CH1 constant domain of the second antigen binding fragment, the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbering according to EU index according to Kabat).

The bispecific antibody does not contain both modifications specified in i) and ii). The constant domains CL and CH1 of the antigen-binding fragment containing the VH/VL exchange are not replaced with each other (ie, they remain unexchanged).

In a more specific embodiment,

i) in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the first antigen binding fragment, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU numbering); or

ii) in the CL constant domain of the second antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the second antigen-binding fragment, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

In one such embodiment, in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the first antigen binding fragment, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

In a further embodiment, in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the first antigen binding fragment, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In a specific embodiment, in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU index Kabat) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In a more specific embodiment, in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is replaced with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced with lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the first antigen binding fragment, the amino acid at position 147 is replaced by glutamic acid (E) (Kabat EU numbering) and the amino acid at position 213 is replaced by glutamic acid (E) (Kabat EU numbering).

In an even more specific embodiment, in the CL constant domain of the first antigen binding fragment, the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbering according to Kabat), and in the constant In the CH1 domain of the first antigen-binding fragment, the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to Kabat's EU index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to Kabat's EU index).

In specific embodiments, if amino acid substitutions according to the above embodiments are made in the CL constant domain and the CH1 constant domain of the first antigen binding fragment, the CL constant domain of the first antigen binding fragment is of the kappa isotype.

Alternatively, amino acid substitutions in accordance with the above embodiments may be made in the CL constant domain and the CH1 constant domain of the second antigen binding fragment instead of the CL constant domain and the CH1 constant domain of the first antigen binding fragment. In specific embodiments, the CL constant domain of the second antigen binding fragment is of the canna isotype.

Accordingly, in one embodiment, in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the second antigen binding fragment, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In a further embodiment, in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the second antigen binding fragment, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In another embodiment, in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the second antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the EU index Kabat) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In one embodiment, in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is replaced with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced with lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding fragment, the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index).

In another embodiment, in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding fragment, the amino acid at position 147 is replaced by glutamic acid (E) (Kabat EU numbering) and the amino acid at position 213 is replaced by glutamic acid (E) (Kabat EU numbering).

To improve heterodimerization of the Fc domain of these asymmetric (heterodimeric) proteins, in one embodiment, in accordance with these aspects of the invention, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) and, optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

In a further embodiment in accordance with these aspects of the invention, in the first subunit of the Fc domain, further the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced cysteine residue), and in the second subunit of the Fc domain, an additional tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering in accordance with the EU index according to Kabat).

Alternative heterodimerization techniques are described below in the section "Fc domains" and are also provided as additional embodiments of the invention.

In a further embodiment in accordance with these aspects of the invention, the Fc domain is a human IgG1 Fc domain.

Fc domains and modifications

In specific embodiments, the bispecific antibody of the invention comprises an Fc domain consisting of first and second subunits. It is understood that the characteristics of the Fc domain described herein in connection with the bispecific antibody can be equally applied to the Fc domain contained in the antibody of the invention.

The Fc domain of a bispecific antibody consists of a pair of polypeptide chains containing the heavy chain domains of the immunoglobulin molecule. For example, the Fc domain of the immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains the CH2 and CH3 constant domains of the IgG heavy chain. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, a bispecific antibody of the invention contains at most one Fc domain.

In one embodiment, the Fc domain of the bispecific antibody is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG ₁ Fc domain. In another embodiment, the Fc domain is an IgG ₄ Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain containing an amino acid substitution at position S228 (Kabat EU numbering), in particular the amino acid substitution S228P. This amino acid substitution reduces the in vivo turnover of Fab arms of IgG ₄ antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further specific embodiment, the Fc domain is a human Fc domain. In a more specific embodiment, the Fc domain is a human IgG ₁ Fc domain.

The Fc domains of the IgG isotype are characterized by different properties based on their interaction with Fc gamma receptors or with the neonatal Fc receptor (FcRn) (see, for example, Vidarsson et al.; Front Immunol 5 (2014) Article 520, 1-17) .

Fc domain modifications promoting heterodimerization

The bispecific antibodies of the invention contain different antigen binding domains that can be fused to one or the other of two Fc domain subunits, such that the two Fc domain subunits are typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of bispecific antibodies when produced recombinantly, it would be advisable to introduce a modification into the Fc domain of a bispecific antibody that facilitates the association of the necessary polypeptides.

Accordingly, in certain embodiments, the Fc domain of a bispecific antibody according to the invention contains a modification that promotes association of the first and second subunits of the Fc domain. The site of the most active protein-protein interaction between the two subunits of the Fc domain of human IgG is the CH3 domain of the Fc domain. Thus, in one embodiment, said modification is located in the CH3 domain of the Fc domain.

There are several approaches for modification in the CH3 domain of the Fc domain in order to stimulate heterodimerization, which are well described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/ 089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3- domain of the second subunit of the Fc domain are constructed in a complementary manner so that each CH3 domain (or the heavy chain containing it) can no longer form a homodimer with itself, but is forced to form a heterodimer with a complementarily designed other CH3 domain (so that heterodimerization of the first and second CH3 domains occurs) domains and there was no formation of homodimers between the first two or two second CH3 domains). These different approaches to improve heavy chain heterodimerization are provided as different alternatives in combination with heavy-light chain modifications (e.g. exchange/replacement of VH and VL in the same binding arm and introducing substitutions of charged amino acids with opposite charges at the CH1/CL interface) in a bispecific antibody, which reduces heavy/light chain mispairing and Bence-Jones by-products.

In a specific embodiment, said modifications to promote association of the first and second Fc domain subunits are a so-called knob-to-cavity modification, comprising a knob modification in one of the two Fc domain subunits and a notch modification in the other. two subunits of the Fc domain.

The "protrusion into recess" technology is described, for example, in US 5731168; US 7695936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, this method involves introducing a bulge ("protrusion") into the contact surface of a first polypeptide and a corresponding cavity ("groove") into the contact surface of a second polypeptide, such that the bulge can be positioned in the cavity so as to promote heterodimer formation and inhibit homodimer formation. Bumps are constructed by replacing small amino acid side chains in the contact surface of the first polypeptide with larger side chains (eg tyrosine or tryptophan). Compensatory cavities of identical or similar size with bulges are created in the contact surface of the second polypeptide by replacing large amino acid side chains with smaller ones (for example, alanine or threonine).

Accordingly, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antibody, an amino acid residue is replaced by an amino acid residue having a larger side chain volume, thereby creating a bulge in the CH3 domain of the first subunit that can be located in a cavity in the CH3 -domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced by an amino acid residue having a smaller side chain volume, thus creating a cavity in the CH3 domain of the second subunit, in which a bulge in the CH3 domain can be located first subunit.

Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

The bulge and cavity can be created by altering the nucleic acid encoding the polypeptides, for example, by site-directed mutagenesis or by peptide synthesis.

In a specific embodiment, in (in the CH3 domain) the first subunit of the Fc domain (the "overhang" subunit), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in (in the CH3 domain) the second subunit of the Fc domain (the "subunit" depressions"), the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbered according to the Kabat EU index).

In a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced by a cysteine residue), and in in the second subunit of the Fc domain, an additional tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering in accordance with the EU index according to Kabat). The addition of two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a specific embodiment, the first Fc domain subunit contains amino acid substitutions S354C and T366W, and the second Fc domain subunit contains amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU numbering).

In a specific embodiment, an antigen binding fragment that binds to a second antigen is fused (optionally through a first antigen binding fragment that binds CCL2 and/or a peptide linker) to a first Fc domain subunit (comprising the overhang modification). Without being limited by theory, fusing an antigen-binding moiety that binds a second antigen, such as an activating T-cell antigen, to a knob-containing Fc domain subunit will (further) minimize the creation of antibodies containing two antigen-binding moieties that bind to an activating T-cell antigen. cellular antigen (due to steric mismatch between two knob-containing polypeptides).

Other CH3 modification techniques for promoting heterodimerization are provided as alternatives in accordance with the invention and are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, an alternative is the heterodimerization approach described in EP 1870459. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between two Fc domain subunits. One preferred embodiment for the bispecific antibody of the invention is the amino acid mutations R409D; K370E in one of the two CH3 domains (Fc domain) and amino acid mutation D399K; E357K in the other of the two CH3 domains of the Fc domain (numbered according to the Kabat EU index).

In another embodiment, the bispecific antibody of the invention contains the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the amino acid mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numbering in accordance with the EU index according to Kabat).

In another embodiment, the bispecific antibody of the invention contains amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the specified bispecific antibody contains amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbering is in accordance with the EU index according to Kabat).

In one embodiment, the heterodimerization approach described in WO 2013/157953 is used as an alternative. In one embodiment, the first CH3 domain contains the amino acid mutation T366K and the second CH3 domain contains the amino acid mutation L351D (Kabat EU numbering). In a further embodiment, the first CH3 domain contains an additional amino acid mutation L351K. In a further embodiment, the second CH3 domain contains an additional amino acid mutation selected from Y349E, Y349D and L368E (in particular L368E) (Kabat EU numbering).

In one embodiment, the heterodimerization approach described in WO 2012/058768 is used as an alternative. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain contains an additional amino acid mutation at position T411, D399, S400, F405, N390 or K392, for example selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numbered according to Kabat EU index). In a further embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain contains the amino acid mutation Y407A, and the second CH3 domain contains the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (Kabat EU numbering).

In one embodiment, the heterodimerization approach described in WO 2011/143545 is alternatively used, for example with an amino acid modification at a position selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index).

In one embodiment, an alternative is to use the heterodimerization approach described in WO 2011/090762, which also uses the lip-to-valve technology described above. In one embodiment, the first CH3 domain contains the T366W amino acid mutation and the second CH3 domain contains the Y407A amino acid mutation. In one embodiment, the first CH3 domain contains the T366Y amino acid mutation and the second CH3 domain contains the Y407T amino acid mutation (Kabat EU numbering).

In one embodiment, the bispecific antibody or Fc domain thereof is of the IgG ₂ subclass, and alternatively the heterodimerization approach described in WO 2010/129304 is used.

In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain includes a modification that mediates the effects of electrostatic interaction, for example, as described in PCT publication WO 2009/089004. In general, this method involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically disadvantageous but heterodimerization is electrostatically advantageous. In one such embodiment, the first CH3 domain contains the amino acid substitution K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain contains the amino acid substitution D399, E356 , D356 or E357 with a positively charged amino acid (eg, lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, and more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (eg, glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain additionally or alternatively comprises an amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering according to Kabat's EU index) .

In a further embodiment, the heterodimerization approach described in WO 2007/147901 is used as an alternative. In one embodiment, the first CH3 domain contains the amino acid mutations K253E, D282K and K322D, and the second CH3 domain contains the amino acid mutations D239K, E240K and K292D (numbered according to the Kabat EU index).

In another embodiment, the heterodimerization approach described in WO 2007/110205 can be used as an alternative.

In one embodiment, the first Fc domain subunit contains amino acid substitutions K392D and K409D, and the second Fc domain subunit contains amino acid substitutions D356K and D399K (Kabat EU index numbering).

As used herein, the term “wild type (WT) IgG or IgG1” with respect to anti-CCL2 bispecific antibodies refers to a bispecific antibody that contains an IgG or IgG1 heavy chain constant region that may contain the modifications/mutations described above to promote heterodimerization, but which does not contains additional Fc domain modifications/mutations that increase or decrease Fc receptor binding and/or effector function, as described below.

Fc domain modifications/mutations that increase or decrease Fc receptor binding and/or effector function:

Modification of bispecific antibodies to CCL2 using sweep technology

The CCL2 bispecific antibodies were modified using sweep technology so that the CCL2 bispecific antibodies could reduce the amount of free CCL2 over long periods of time to allow long-lasting biological effects such as anti-cancer efficacy in vivo.

The concept of sweeping is described, for example, in Igawa et al, Immunological Reviews 270 (2016) 132-151, WO 2012/122011, WO 2016/098357 and WO 2013/081143, which are incorporated herein by reference.

The present invention provides methods for facilitating antibody-mediated uptake of an antigen into a cell by reducing the antigen-binding activity of the above-described antibody in the acidic pH range to less than its antigen-binding activity in the neutral pH range; and this facilitates the uptake of the antigen into the cell. The present invention also provides methods for facilitating antibody-mediated uptake of an antigen into a cell that rely on altering at least one amino acid in the antigen binding domain of the above-described antibody to facilitate uptake of the antigen into a cell. The present invention also provides methods for facilitating antibody-mediated uptake of an antigen into a cell, which are based on the substitution of at least one amino acid with histidine or the insertion of at least one histidine into the antigen binding domain of the above-described antibody, which facilitates the uptake of the antigen into the cell.

As used herein, “antibody-mediated uptake of antigens into cells” means that the uptake of antigens into cells occurs through endocytosis. As used herein, “facilitate cellular uptake” means that the rate of intracellular uptake of antigen-bound antibody into plasma is increased and/or the amount of return of absorbed antigen to plasma is decreased. This means that the rate of cellular uptake is increased relative to the antibody until human FcRn binding activity increases in the neutral pH range or until human FcRn binding activity increases and the antigen binding activity (binding ability) of the antibody in the acidic pH range decreases to less than its antigen binding activity in a neutral pH range. The increase in rate occurs preferentially compared to intact human IgG and more preferably compared to intact human IgG. Thus, in the present invention, whether the uptake of antigen by cells is facilitated can be assessed based on the increase in the rate of uptake of antigen by cells. The rate of antigen uptake by cells can be calculated, for example, by monitoring the decrease over time in the concentration of antigen in a culture medium containing human FcRn-expressing cells after adding antigen and antibody to the medium, or by monitoring the rate of antigen uptake over time by human FcRn-expressing cells. By using the methods of the present invention to increase the rate of antibody-mediated uptake of antigen into cells, for example, it is possible to increase the rate of elimination of antigen from plasma by administering antibodies. Thus, whether antibody-mediated cellular uptake of antigen is facilitated can also be assessed, for example, by examining whether the rate of elimination of antigen from plasma is increased or whether total plasma antigen concentration is decreased upon administration of the antibody.

As used herein, “total plasma antigen concentration” means the total concentration of antibody-bound antigen and non-antibody-bound antigen, or “plasma free antigen concentration,” which is the concentration of non-antibody-bound antigen. Methods for measuring “total plasma antigen concentration” or “free plasma antigen concentration” are well known in the art and are described later herein.

As used herein, “intact human IgG” (or “wild-type (WT) human IgG”) means unmodified (except for potential modifications for heterodimerization described above) human IgG and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "intact human IgG" provided that it can bind to human FcRn in the acidic pH range. Preferably, the "intact human IgG" may be human IgG1.

The present invention also provides methods for increasing the number of antigens to which one antibody can bind. More specifically, the present invention provides methods for increasing the number of antigens to which a single antibody having human FcRn binding activity in an acidic pH range can bind by increasing the binding activity of a human FcRn antibody in a neutral pH range. The present invention also provides methods for increasing the number of antigens to which a single antibody having human FcRn binding activity in the acidic pH range can bind by changing at least one amino acid in the human FcRn binding domain of the antibody.

The present invention provides methods for facilitating antibody-mediated uptake of antigen into cells. More specifically, the present invention provides methods for facilitating the uptake of antigen into cells by an antibody having human FcRn binding activity in the acidic pH range, which is based on increasing the binding activity of the human FcRn antibody in the neutral pH range. The present invention also provides methods for improving the uptake of antigen into cells by an antibody having human FcRn binding activity in the acidic pH range, which is based on changing at least one amino acid in the human FcRn binding domain of the antibody.

The present invention also provides methods for facilitating the uptake of an antigen into cells by an antibody having human FcRn binding activity in the acidic pH range, which is based on the use of a human FcRn binding domain containing an amino acid sequence with a different amino acid replacing at least one amino acid selected from the amino acids at positions 237 , 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315 , 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering), in the Fc domain of the parental IgG binding human FcRn domain containing the Fc domain of the parent IgG.

The present invention also provides methods for facilitating antibody-mediated uptake of an antigen into a cell by reducing the antigen-binding activity of the above-described antibody in the acidic pH range to less than its antigen-binding activity in the neutral pH range; and this facilitates the uptake of the antigen into the cell. The present invention also provides methods for facilitating antibody-mediated uptake of an antigen into a cell that rely on altering at least one amino acid in the antigen binding domain of the above-described antibody to facilitate uptake of the antigen into a cell. The present invention also provides methods for facilitating antibody-mediated uptake of an antigen into a cell, which are based on the substitution of at least one amino acid with histidine or the insertion of at least one histidine into the antigen binding domain of the above-described antibody, which facilitates the uptake of the antigen into the cell.

As used herein, “antibody-mediated uptake of antigens into cells” means that the uptake of antigens into cells occurs through endocytosis. As used herein, “facilitate cellular uptake” means that the rate of intracellular uptake of antigen-bound antibody into plasma is increased and/or the amount of return of absorbed antigen to plasma is decreased. This means that the rate of cellular uptake is increased relative to the antibody until human FcRn binding activity increases in the neutral pH range or until human FcRn binding activity increases and the antigen binding activity (binding ability) of the antibody in the acidic pH range decreases to less than its antigen binding activity in a neutral pH range. The increase in rate occurs preferentially compared to intact human IgG and more preferably compared to intact human IgG. Thus, in the present invention, whether the uptake of antigen by cells is facilitated can be assessed based on the increase in the rate of uptake of antigen by cells. The rate of antigen uptake by cells can be calculated, for example, by monitoring the decrease over time in the concentration of antigen in a culture medium containing human FcRn-expressing cells after adding antigen and antibody to the medium, or by monitoring the rate of antigen uptake over time by human FcRn-expressing cells. By using the methods of the present invention to increase the rate of antibody-mediated uptake of antigen into cells, for example, it is possible to increase the rate of elimination of antigen from plasma by administering antibodies. Thus, whether antibody-mediated cellular uptake of antigen is facilitated can also be assessed, for example, by examining whether the rate of elimination of antigen from plasma is increased or whether total plasma antigen concentration is decreased upon administration of the antibody.

As used herein, “total plasma antigen concentration” means the total concentration of antibody-bound antigen and non-antibody-bound antigen, or “plasma free antigen concentration,” which is the concentration of non-antibody-bound antigen. Methods for measuring “total plasma antigen concentration” or “free plasma antigen concentration” are well known in the art and are described later herein.

As used herein, “intact human IgG” (or “wild-type IgG”) means unmodified human IgG (excluding potential modifications for heterodimerization described above) and is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be used as "intact human IgG" provided that it can bind to human FcRn in the acidic pH range. Preferably, the "intact human IgG" may be human IgG1.

As used herein, “parental IgG” means an unmodified IgG that is subsequently modified to create a variant, provided that the modified variant of the parental IgG can bind to human FcRn in the acidic pH range (hence, the parental IgG does not need to have human FcRn binding activity under acidic conditions). The parent IgG may be a naturally occurring IgG or a variant or engineered version of a naturally occurring IgG. Parental IgG may refer to the polypeptide itself, compositions that contain the parental IgG, or the amino acid sequence that encodes it. It should be noted that "parental IgG" includes known commercial, recombinantly produced IgG, described below. The origin of the "parental IgG" is not restricted, and it can be obtained from any non-human animal or human organism. Preferably, the organism is selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel and non-human primate. In another embodiment, the “parental IgG” may also be obtained from a cynomolgus monkey, marmoset, rhesus monkey, chimpanzee, or human. Preferably, the “parent IgG” is derived from human IgG1, but is not limited to a particular class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can respectively be used as the “parent IgG”. Likewise, any class or subclass of IgG from any of the above-described organisms may preferably be used as the “parent IgG.” Examples of a variant or engineered version of a naturally occurring IgG are described in Curr Opin Biotechnol. 2009 Dec; 20(6): 685-91, Curr Opin Immunol. 2008 Aug; 20(4): 460-70, Protein Eng Des Sel. 2010 Apr; 23(4): 195-202, WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338, but not limited to them.

The present invention also provides methods for increasing the ability to eliminate plasma antigen by administering antibodies. In the present invention, the expression “methods for enhancing the ability to eliminate plasma antigen” is synonymous with the expression “methods for enhancing the ability of an antibody to eliminate antigen from plasma.” More specifically, the present invention provides methods for increasing the plasma antigen elimination ability of an antibody having human FcRn binding activity in an acidic pH range by increasing the binding activity of a human FcRn antibody in a neutral pH range. The present invention also provides methods for increasing the plasma antigen elimination ability of an antibody having human FcRn binding activity in the acidic pH range, which is based on changing at least one amino acid in the human FcRn binding domain of the antibody.

The present invention also provides methods for enhancing the plasma antigen elimination ability of an antibody having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain containing an amino acid sequence substituting at least one amino acid selected from amino acids at positions 237, 238 , 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317 , 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering), in the Fc domain of the parental IgG human FcRn binding domain, containing the Fc domain of the parent IgG, a different amino acid.

The present invention also provides methods for increasing the plasma antigen elimination ability of an antibody by reducing the antigen binding activity in the acidic pH range of the above-described antibody with improved plasma antigen elimination ability compared to the antigen binding activity in the neutral pH range. The present invention also provides methods for improving the plasma antigen elimination ability of an antibody by changing at least one amino acid in the antigen binding domain of the above-described antibody with improved plasma antigen elimination ability. The present invention also provides methods for increasing the plasma antigen elimination ability of an antibody by replacing at least one amino acid with histidine or inserting at least one histidine in the antigen binding domain of the above-described antibody with improved plasma antigen elimination ability.

As used herein, “plasma antigen elimination ability” means the ability to eliminate antigen from plasma upon administration or secretion of antibodies in vivo. Thus, as used herein, “increased plasma antigen elimination capacity” means that there is an increase in the rate of elimination of antigen from plasma after administration of an antibody compared to the rate before the human FcRn binding activity of the antibody increases in the neutral pH range or before the human FcRn binding activity increases and simultaneous reduction of antigen-binding activity in the acidic pH range to less than in the neutral pH range. Increased activity of an antibody to eliminate antigen from plasma can be assessed, for example, by administering the soluble antigen and antibody in vivo and measuring the plasma concentration of the soluble antigen after administration. When the concentration of a soluble antigen in plasma after administration of a soluble antigen and antibody is reduced by increasing the human FcRn binding activity of the antibody in the neutral pH range or increasing its human FcRn binding activity and simultaneously reducing its antigen binding activity in the acidic pH range to less than in the neutral pH range , we can assume that there is an increase in the ability of the antibody to eliminate plasma antigen. The form of soluble antigen can be antibody-bound antigen or non-antibody-bound antigen, whose concentration can be defined as the “plasma concentration of antibody-bound antigen” and the “plasma concentration of non-antibody-bound antigen,” respectively (the latter being synonymous with “plasma concentration of free antigen "). Since "total plasma antigen concentration" means the total concentration of antibody-bound antigen and non-antibody-bound antigen, or "plasma free antigen concentration", which is the concentration of non-antibody-bound antigen, soluble antigen concentration can be defined as "total plasma antigen concentration " Methods for measuring “total plasma antigen concentration” or “free plasma antigen concentration” are well known in the art and are described later herein.

The present invention also provides methods for improving the pharmacokinetics of antibodies. More specifically; The present invention provides methods for improving the pharmacokinetics of an antibody having human FcRn binding activity in the acidic pH range by increasing the human FcRn binding activity of the antibody in the neutral pH range. In addition, the present invention provides methods for improving the pharmacokinetics of an antibody having human FcRn binding activity in the acidic pH range by altering at least one amino acid in the human FcRn binding domain of the antibody.

The present invention also provides methods for improving the pharmacokinetics of an antibody having human FcRn binding activity in the acidic pH range by using a human FcRn binding domain comprising an amino acid sequence substituting a different amino acid for at least one amino acid selected from amino acids at positions 237, 238, 239 , 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325 , 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434 and 436 (EU numbering), in the Fc domain of the parental IgG human FcRn binding domain containing the Fc -IgG domain.

The plasma concentration of free antigen not bound to an antibody, or the ratio of free antigen concentration to total concentration, can be determined by methods known to those skilled in the art, for example, the method described in Pharm Res. 2006 Jan; 23(I): 95-103. Alternatively, when an antigen exhibits a particular function in vivo, whether the antigen is associated with an antibody that neutralizes the function of the antigen (antagonist molecule) can be assessed by examining whether the function of the antigen is neutralized. Whether neutralization of antigen function occurs can be assessed by assaying an in vivo marker that reflects antigen function. Whether an antigen is associated with an antibody that activates the function of the antigen (agonist molecule) can be assessed by analyzing an in vivo marker that reflects the function of the antigen.

Determination of the plasma concentration of free antigen and the ratio of the amount of free antigen in plasma to the total amount of antigen in plasma, in vivo marker analysis and the like are not particularly limited; in this case, the analysis is preferably carried out after a certain period of time has passed after administration of the antibody. In the present invention, the period after administration of the antibody is not particularly limited; those skilled in the art can determine the appropriate period depending on properties and the like. injected antibody. Such periods include, for example, one day after antibody administration, three days after antibody administration, seven days after antibody administration, 14 days after antibody administration, and 28 days after antibody administration. As used herein, “plasma antigen concentration” means “total plasma antigen concentration,” which is the total concentration of antibody-bound antigen and non-antibody-bound antigen, or “plasma free antigen concentration,” which is the concentration of non-antibody-bound antigen.

The total antigen concentration in plasma can be reduced by administering an antibody of the present invention by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or even more compared to the administration of a reference antibody, containing an intact human IgG Fc domain as an FcRn binding domain, or compared to the case where the antigen binding domain molecule of the present invention is not introduced.

In another aspect, the invention provides bispecific antibodies to CCL2 that exhibit pH-dependent binding characteristics. As used herein, the expression “pH-dependent binding” means that the antibody exhibits “reduced binding to CCL2 at acidic pH compared to binding at neutral pH” (for the purposes of the present invention, both expressions can be used interchangeably). For example, antibodies with “pH-dependent binding characteristics” include antibodies that bind to CCL2 with greater affinity at neutral pH than at acidic pH. In certain embodiments, the bispecific antibodies of the present invention bind to CCL2 with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times greater affinity at neutral pH than at acidic pH. In some embodiments, the antibodies bind to CCL2 with greater affinity at pH 7.4 than at pH 5.8. In additional embodiments, the antibodies bind to CCL2 with at least 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95, 100, 200, 400, 1000, 10000 or more times greater affinity at pH 7.4 than at pH 5.8.

When the antigen is a soluble protein, binding of the antibody to the antigen may result in prolongation of the plasma half-life of the antigen (i.e., decreased clearance of the antigen from plasma) because the antibody may have a longer plasma half-life than the antigen itself and may serve as a carrier for antigen. This occurs due to recycling of the antigen-antibody complex by FcRn through the endosomal pathway into the cell (Roopenian, Nat. Rev. Immunol. 7(9): 715-725 (2007)). An antibody with pH-dependent binding characteristics that binds its antigen in a neutral extracellular environment, releasing the antigen into acidic endosomal compartments upon entry into the cell, is expected to have superior properties in terms of neutralization and antigen clearance compared to an antibody that binds in a pH-independent manner (Igawa et al., Nature Biotechnol. 28(11):1203-1207 (2010); Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009/125825).

The “affinity” of an antibody for CCL2 for the purposes of the present invention is expressed in terms of the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value for an antibody to bind to its antigen, the weaker the binding affinity for that particular antigen. Accordingly, as used herein, the expression “greater affinity at neutral pH than at acidic pH” (or the equivalent expression “pH-dependent binding”) means that the KD of antibody binding to CCL2 at acidic pH is greater than the KD of antibody binding to CCL2 at acidic pH. neutral pH. For example, in the context of the present invention, an antibody is considered to bind to CCL2 with greater affinity at neutral pH than at acidic pH if the KD of antibody binding to CCL2 at acidic pH is at least 2 times greater than the KD of antibody binding to CCL2 at neutral pH pH. Thus, the present invention includes antibodies that bind to CCL2 at an acidic pH with a KD that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the KD of antibody binding to CCL2 at neutral pH. In another embodiment, the KD value of the antibody at neutral pH may be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M or less. In another embodiment, the KD value of the antibody at an acidic pH may be 10-9 M, 10-8 M, 10-7 M, 10-6 M or more.

In additional embodiments, an antibody is considered to bind with greater affinity at neutral pH than at acidic pH if the KD of the antibody binding to CCL2 at pH 5.8 is at least 2 times greater than the KD of the antibody binding to CCL2 at pH 7. 4. In some embodiments, antibodies are provided that bind to CCL2 at pH 5.8 with a KD that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60. 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10,000 or more times the KD of antibody binding to CCL2 at pH 7.4. In another embodiment, the KD value of the antibody at pH 7.4 may be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10-9 M, 10-8 M, 10-7 M, 10-6 M or more.

The binding properties of an antibody to a particular antigen can also be expressed in terms of the kd of the antibody, the kd of the antibody refers to the dissociation rate constant of the antibody to a particular antigen and is expressed in reciprocal seconds (i.e. s-1). An increase in kd value means weaker binding of the antibody to its antigen. Therefore, the present invention includes antibodies that bind to CCL2 with a higher kd value at acidic pH than at neutral pH. The present invention includes antibodies that bind to CCL2 at an acidic pH with a kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65. 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times the kd of antibody binding to CCL2 at neutral pH. In another embodiment, the kd value of the antibody at neutral pH may be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s or less. In another embodiment, the kd value of the antibody at an acidic pH may be 10-3 1/s, 10-2 1/s, 10-1 1/s or more. The invention also includes antibodies that bind to CCL2 with a greater kd value at pH 5.8 than at pH 7.4. The invention includes antibodies that bind to CCL2 at pH 5.8 with a kd that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times greater than the kd of antibody binding to CCL2 at pH 7.4. In another embodiment, the kd value of the antibody at pH 7.4 may be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s or less . In another embodiment, the kd value of the antibody at pH 5.8 may be 10-3 1/s, 10-2 1/s, 10-1 1/s or more.

In certain cases, “reduced binding to CCL2 at acidic pH relative to its binding at neutral pH” is expressed as the ratio of the KD value of antibody binding to CCL2 at acidic pH to the KD value of antibody binding to CCL2 at neutral pH (or vice versa). For example, for purposes of the present invention, an antibody may be considered to exhibit “reduced binding to CCL2 at acidic pH compared to its binding at neutral pH” if the antibody exhibits an acid/neutral KD ratio of 2 or more. In certain embodiments, the pH 5.8/pH 7.4 KD ratio for the anti-CCL2 antibody of the present invention is 2 or more. In certain exemplary embodiments, the acid/neutral KD ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at neutral pH may be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M or less. In another embodiment, the KD value of the antibody at an acidic pH may be 10-9 M, 10-8 M, 10-7 M, 10-6 M or more. In additional cases, an antibody may be considered to exhibit “reduced binding to CCL2 at acidic pH compared to its binding at neutral pH” if the antibody exhibits a pH 5.8/pH 7.4 KD ratio of 2 or more. In certain exemplary embodiments, the pH 5.8/pH 7.4 KD ratio for an antibody may be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at pH 7.4 may be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M or less. In another embodiment, the KD value of the antibody at pH 5.8 may be 10-9 M, 10-8 M, 10-7 M, 10-6 M or more.

In certain cases, “reduced binding to CCL2 at acidic pH relative to its binding at neutral pH” is expressed as the ratio of the kd value of antibody binding to CCL2 at acidic pH to the kd value of antibody binding to CCL2 at neutral pH (or vice versa). For example, for purposes of the present invention, an antibody may be considered to exhibit “reduced binding to CCL2 at acidic pH compared to its binding at neutral pH” if the antibody exhibits an acid/neutral kd ratio of 2 or more. In certain exemplary embodiments, the pH 5.8/pH 7.4 kd ratio for an antibody of the present invention is 2 or more. In certain exemplary embodiments, the acid/neutral kd ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH may be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s or less. In another embodiment, the kd value of the antibody at an acidic pH may be 10-3 1/s, 10-2 1/s, 10-1 1/s or more. In certain exemplary embodiments, the pH 5.8/pH 7.4 kd ratio for an antibody of the present invention may be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at pH 7.4 may be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s or less . In another embodiment, the kd value of the antibody at pH 5.8 may be 10-3 1/s, 10-2 1/s, 10-1 1/s or more.

As used herein, the expression “acidic pH” means a pH between 4.0 and 6.5. The expression "acid pH" includes any pH value from 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6, 2, 6.3, 6.4 and 6.5. In specific aspects, the "acid pH" is 5.8.

As used herein, the expression “neutral pH” means a pH between 6.7 and about 10.0. The expression "neutral pH" includes any pH value from 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8, 9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 and 10.0. In specific aspects, the "neutral pH" is 7.4.

The KD values and kd values reported herein can be determined using a biosensor based on the phenomenon of surface plasma resonance to characterize the antibody-antigen interaction. KD values and kd values can be determined at 25 degrees C or 37 degrees C.

In a further aspect, the invention provides a bispecific antibody to CCL2 that forms an immune complex (ie, antigen-antibody complex) with CCL2. In certain embodiments, two or more bispecific antibodies to CCL2 bind to two or more CCL2 molecules to form an immune complex. This is possible because CCL2 exists as a homodimer containing two CCL2 molecules, whereas the antibody has two antigen-binding sites.

In general, when two or more antibodies form an immune complex with two or more antigens, the resulting immune complex can bind strongly to Fc receptors on the surface of cells due to avidity effects through the Fc regions of the antibodies in the complex, and can then be taken up by the cell with greater efficiency. Thus, the above-mentioned anti-CCL2 antibody capable of forming an immune complex containing two or more anti-CCL2 antibodies and two or more CCL2 molecules can lead to rapid clearance of CCL2 from plasma in a living body by strongly binding to Fc receptors due to avidity effects.

In addition, it is believed that an antibody with pH-dependent binding characteristics will have superior properties in terms of neutralization and antigen clearance compared to an antibody that binds in a pH-independent manner (Igawa et al., Nature Biotech. 28(11): 1203 -1207 (2010); Devanaboyina et al. mAbs 5(6): 851-859 (2013); Therefore, it is expected that an antibody having both of the above properties, that is, an antibody that has pH-dependent binding characteristics and that forms an immune complex containing two or more antibodies with two or more antigens, will have even more superior properties against a strong increase in the elimination of antigens from plasma (WO 2013/081143).

In one aspect, the invention provides polypeptides comprising variant Fc regions with increased FcgammaRIIb binding activity, comprising at least two amino acid changes comprising: (a) a change in one amino acid at position 236 and (b) a change in at least one amino acid at at least one item selected from the group consisting of: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334 and 396 according to EU numbering.

In one aspect, the invention provides polypeptides comprising a variant Fc region with increased FcgammaRIIb binding activity comprising an amino acid change at position 236 according to EU numbering.

In one aspect, the invention provides polypeptides comprising a variant Fc region with increased FcgammaRIIb binding activity comprising at least two amino acid changes comprising: (a) a change in one amino acid at position 236 and (b) a change in at least one amino acid at at least one item selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396 according to EU numbering. In a further embodiment, the variant Fc region comprises an amino acid change at at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330 and 396 according to EU numbering. In a further embodiment, the variant Fc region comprises an amino acid change at at least one position selected from the group consisting of: 268, 295, 326 and 330 according to EU numbering.

In another aspect, the invention provides polypeptides containing variant Fc regions with increased FcgammaRIIb binding activity, containing amino acid changes according to any of the following options (1)-(37): (1) positions 231, 236, 239, 268 and 330; (2) positions 231, 236, 239, 268, 295 and 330; (3) positions 231, 236, 268 and 330; (4) positions 231, 236, 268, 295 and 330; (5) positions 232, 236, 239, 268, 295 and 330; (6) positions 232, 236, 268, 295 and 330; (7) positions 232, 236, 268 and 330; (8) positions 235, 236, 268, 295, 326 and 330; (9) positions 235, 236, 268, 295 and 330; (10) positions 235, 236, 268 and 330; (11) positions 235, 236, 268, 330 and 396; (12) positions 235, 236, 268 and 396; (13) positions 236, 239, 268, 295, 298 and 330; (14) positions 236, 239, 268, 295, 326 and 330; (15) positions 236, 239, 268, 295 and 330; (16) positions 236, 239, 268, 298 and 330; (17) positions 236, 239, 268, 326 and 330; (18) positions 236, 239, 268 and 330; (19) positions 236, 239, 268, 330 and 396; (20) positions 236, 239, 268 and 396; (21) positions 236 and 268; (22) positions 236, 268 and 295; (23) positions 236, 268, 295, 298 and 330; (24) positions 236, 268, 295, 326 and 330; (25) positions 236, 268, 295, 326, 330 and 396; (26) positions 236, 268, 295 and 330; (27) positions 236, 268, 295, 330 and 396; (28) positions 236, 268, 298 and 330; (29) positions 236, 268, 298 and 396; (30) positions 236, 268, 326 and 330; (31) positions 236, 268, 326, 330 and 396; (32) positions 236, 268 and 330; (33) positions 236, 268, 330 and 396; (34) positions 236, 268 and 396; (35) positions 236 and 295; (36) positions 236, 330 and 396; and (37) positions 236 and 396 according to EU numbering.

In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity comprises at least one amino acid selected from the group consisting of: (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (6) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) Asp at position 233; (d) Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) Asp, Tyr at position 237; (i) Glu, Ile, Met, Gln, Tyr at position 238; (j) Ile, Leu, Asn, Pro, Val in position 239; (k) Ile at position 264; (m) Phe at position 266; (m) Ala, His, Leu at position 267; (p) Asp, Glu at position 268; (p) Asp, Glu, Gly at position 271; (c) Leu at position 295; (t) Leu at position 298; (y) Glu, Phe, Ile, Leu at position 325; (f) Thr at position 326; (x) Ile, Asn at position 327; (ts) Thr at position 328; (w) Lys, Arg at position 330; (u) Glu at position 331; (e) Asp at position 332; (u) Asp, Ile, Met, Val, Tyr in position 334; and (i) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396; according to EU numbering.

In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains at least one amino acid change (eg, substitution) selected from the group consisting of: (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (e) Asp, Glu at position 268; (g) Leu at position 295; (i) Leu at position 298; (k) Thr at position 326; (l) Lys, Arg at position 330; and (m) Lys, Met at position 396; according to EU numbering. In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains the following amino acid changes (eg, substitutions): Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396; according to EU numbering. In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains the following amino acid changes (eg, substitutions): Asn at position 236, Asp at position 268, and Lys at position 330; according to EU numbering. In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains the following amino acid changes (eg, substitutions): Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330; according to EU numbering. In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains the following amino acid changes (eg, substitutions): Thr at position 236, Asp at position 268, and Lys at position 330; according to EU numbering. In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains the following amino acid changes (eg, substitutions): Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330; according to EU numbering. In a further embodiment, the variant Fc region with increased FcgammaRIIb binding activity contains the following amino acid changes (eg, substitutions): Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at positions 330; according to EU numbering.

In another aspect, the invention provides isolated polypeptides containing variant Fc regions with an increased isoelectric point (pI). In certain embodiments, a variant Fc region described herein contains at least two amino acid changes in the parent Fc region. In certain embodiments, each of the amino acid changes increases the isoelectric point (pI) of the variant Fc region compared to the parent Fc region. They are based on the discovery that elimination of antigen from plasma can be stimulated by an antibody whose pI has been increased by modification of at least two amino acid residues, for example when the antibody is administered in vivo.

In the present invention, pI may be either a theoretically determined pI or an experimentally determined pI. The pI value can be determined, for example, using isoelectric focusing, known to those skilled in the art. The theoretical pI value can be calculated, for example, using software for analyzing gene and amino acid sequences (Genetyx, etc.).

In one embodiment, the pI value may be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, by at least 0.6, 0.7, 0.8, 0.9 or more, by at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more than or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more compared to the value before modification.

In certain embodiments, the pI-raising amino acid may be located on the surface of the variant Fc region. In the present invention, an amino acid that may be on the surface generally refers to an amino acid residue located on the surface of the polypeptide constituting the variant Fc region. An amino acid residue located on the surface of a polypeptide refers to an amino acid residue whose side chain can contact solvent molecules (which in general are predominantly water molecules). However, the side chain does not have to be in complete contact with the solvent molecules, and even when part of the side chain is in contact with the solvent molecules, an amino acid is defined as a “surface-exposed amino acid residue.” An amino acid residue located on the surface of a polypeptide also includes amino acid residues located close to the surface, and therefore its electrical charge can be influenced by another amino acid residue whose side chain is even partially in contact with solvent molecules. Those skilled in the art can create a homology model of a polypeptide, for example, using commercially available software. Alternatively, methods known to those skilled in the art, such as x-ray crystallography, can be used. Amino acid residues that may be present on the surface are determined, for example, using coordinates from a three-dimensional model using a computer program such as the InsightII program (Accelrys). Surface sites can be determined using algorithms known in the art (eg, Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548- 558 (1983)). Surface sites can be determined using software suitable for protein modeling and 3D structure information. Software available for such purposes includes, for example, SYBYL Biopolymer Module software (Tripos Associates). When the algorithm involves user input of a size parameter, the probe "size" used in the calculations can be set to approximately 1.4 angstroms or less in radius. In addition, methods for determining surface areas using personal computer software, have been described in Pacios (Comput. Chem. 18(4):377-386 (1994); J. Mol. Model. 1:46-53 (1995)) Based on information such as described above, appropriate amino acids can be selected. residues located on the surface of the polypeptide that constitutes the variant Fc region.

In certain embodiments, the polypeptide contains both a variant Fc region and an antigen binding domain. In additional embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in the biological fluids (eg, plasma, interstitial fluid, lymph fluid, ascites fluid, and pleural fluid) of the subjects. The antigen may also be a membrane antigen.

In additional embodiments, the antigen binding activity of the antigen binding domain varies depending on ion concentration conditions. In one embodiment, the ion concentration is not particularly limited and refers to the hydrogen ion concentration (pH) or metal ion concentration. As used herein, metal ions refer to ions of Group I elements excluding hydrogen, such as alkali metals and copper group elements, Group II elements, such as alkaline earth metals and zinc group elements, Group III elements excluding boron, Group IV elements excluding carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI and VII, and metal elements such as antimony, bismuth and polonium. In the present invention, the metal ions include, for example, calcium ion, as described in WO 2012/073992 and WO 2013/125667. In one embodiment, “ion concentration conditions” may be conditions that focus on the difference in the biological behavior of the antigen binding domain between a low ion concentration and a high ion concentration. In addition, the expression “the antigen binding activity of the antigen binding domain changes depending on the ion concentration conditions” means that the antigen binding activity of the antigen binding domain changes between low ion concentration and high ion concentration (such an antigen binding domain is referred to herein as “ion concentration dependent antigen binding domain”). . The antigen-binding activity of the antigen-binding domain under conditions of high ion concentration may be higher (stronger) or lower (weaker) than under conditions of low ion concentration. In one embodiment, ion concentration-dependent antigen binding domains (such as pH-dependent antigen binding domains or calcium ion concentration-dependent antigen binding domains) can be prepared by known methods, for example, those described in WO 2009/125825, WO 2012/073992 and WO 2013/046722 .

In the present invention, the antigen binding activity of the antigen binding domain under high calcium ion concentration conditions can be higher than under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited, but may be a concentration selected between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM, between 400 μM and 1 mM or between 500 μM and 2.5 mM, which is preferably close to the concentration of calcium ions in plasma (blood) in vivo. Here, the low concentration of calcium ions is not particularly limited, but may be a concentration selected between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, between 1 μM and 5 µM or between 2 µM and 4 µM, which is preferably close to the calcium ion concentration in early endosomes in vivo.

In one embodiment, the ratio between the antigen binding activity under low calcium ion concentration conditions and high calcium ion concentration conditions is not limited, but the ratio of the dissociation constant (KD) under low calcium ion concentration conditions to the KD under high calcium ion concentration conditions, i.e. KD (low calcium ion concentration condition)/KO (high calcium ion concentration condition) is 2 or more, 10 or more, or 40 or more. The upper limit of the ratio may be 400, 1000 or 10000, provided that such antigen binding domain can be obtained by techniques known to those skilled in the art. Alternatively, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under conditions of low concentration of calcium ions to kd under conditions of high concentration of calcium ions, i.e. kd (low calcium ion concentration conditions)/kd (high calcium ion concentration conditions) is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the ratio may be 50, 100 or 200, provided that the antigen binding domain can be prepared based on the general technical knowledge of those skilled in the art.

In the present invention, the antigen binding activity of the antigen binding domain under conditions of low hydrogen ion concentration (neutral pH) can be higher than under conditions of high hydrogen ion concentration (acidic pH). The acidic pH may be, for example, a pH selected from pH 4.0 to pH 6.5, selected from pH 4.5 to pH 6.5, selected from pH 5.0 to pH 6.5, or selected from pH 5.5 to pH 6.5, which is preferably close to the in vivo pH in early endosomes. The acidic pH may also be pH 5.8 or pH 6.0. In specific embodiments, the acidic pH is pH 5.8. Here, the neutral pH may be, for example, a pH selected from pH 6.7 to pH 10.0, selected from pH 6.7 to pH 9.5, selected from pH 7.0 to pH 9.0 or selected from pH 7.0 to pH 8.0, which is preferably close to the in vivo pH in plasma (blood). Neutral pH may also be pH 7.4 or pH 7.0. In specific embodiments, the neutral pH is pH 7.4.

In one embodiment, the ratio between antigen binding activity under acidic pH conditions and neutral pH conditions is not limited, but the ratio of the dissociation constant (KD) under acidic pH conditions to the KD under neutral pH conditions, i.e. KD (acidic pH conditions)/KD (neutral pH conditions) is 2 or more, 10 or more, or 40 or more. The upper limit of the ratio may be 400, 1000 or 10000, provided that such antigen binding domain can be obtained by techniques known to those skilled in the art. Alternatively, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions to kd under neutral pH conditions, i.e. kd (acidic pH conditions)/kd (neutral pH conditions) is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the ratio may be 50, 100 or 200, provided that the antigen binding domain can be prepared based on the general technical knowledge of those skilled in the art.

In one embodiment, for example, at least one amino acid residue is replaced by an amino acid residue with a side chain pKa of 4.0 to 8.0 and/or at least one amino acid residue with a side chain pKa of 4.0 to 8.0 is inserted into the antigen binding domain, as described in WO 2009/125825. An amino acid may be substituted and/or inserted at any site provided that the antigen binding activity of the antigen binding domain becomes weaker under acidic pH conditions than under neutral pH conditions compared to the activity before the substitution or insertion was made. When the antigen binding domain contains a variable region or CDR, the region may be within the variable region or CDR. The number of amino acid residues that are substituted or inserted can be appropriately determined by those skilled in the art; and this number may be one or more. Amino acids with a side chain pKa of 4.0-8.0 can be used to alter the antigen binding activity of the antigen binding domain depending on hydrogen ion concentration conditions. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and non-natural amino acids such as histidine analogues (US 2009/0035836), m-NO2-Tyr (pKa 7.45), 3. 5-Br2-Tyr (pKa 7.21) and 3,5-12-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). Amino acids with a side chain pKa of 6.0 to 7.0 can also be used, which include, for example, His (H).

In another embodiment, preferred antigen binding domains for a variant Fc region with increased pI are described, which can be obtained by methods described in WO 2016/125495 and WO 2017/046994.

In certain embodiments, the increased pI variant Fc region contains at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 according to EU numbering.

In additional embodiments, the increased pI variant Fc region contains at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413 according to EU numbering.

In another aspect, the invention provides polypeptides containing variant Fc regions with increased pI, containing amino acid changes according to any of the following options (1)(10): (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) items 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) items 402 and 413; according to EU numbering.

In one aspect, the invention provides polypeptides comprising variant Fc regions with increased FcgammaRIIb binding activity and increased pI, comprising at least three amino acid changes, including: (a) a change in at least one amino acid at at least one position selected from group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332 , 334 and 396 in accordance with EU numbering, and (b) changes in at least two amino acids at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 according to EU numbering.

In one aspect, the invention provides polypeptides comprising variant Fc regions with increased FcgammaRIIb binding activity and increased pI, and which contain at least three amino acid changes, including: (a) a change in at least one amino acid at at least one position, selected from the group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326, 330 and 396 according to EU numbering, and (b) a change in at least two amino acids in at least two positions , selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402 and 413 according to EU numbering.

In another aspect, the invention provides polypeptides containing variant Fc regions with increased FcgammaRIIb binding activity and increased pI, containing amino acid changes according to any of the following options (1)(9): (1) positions 235, 236, 268, 295, 311 , 326, 330 and 343; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311, 330 and 413; (4) positions 236, 268, 311, 330, 396 and 399; (5) positions 236, 268, 311, 330 and 343; (6) positions 236, 268, 311, 330, 343 and 413; (7) positions 236, 268, 311, 330, 384 and 413; (8) positions 236, 268, 311, 330 and 413; and (9) positions 236, 268, 330, 396, 400 and 413; according to EU numbering.

In one aspect, the invention provides polypeptides comprising variant Fc regions with increased FcgammaRIIb binding activity and increased pI, comprising at least three amino acid changes, including: (a) a change in at least one amino acid at at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307 and 330, and (b) changes in at least two amino acids at at least two positions selected from the group consisting of: 285, 311, 312, 315 , 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 according to EU numbering. In further embodiments, the polypeptides contain at least two amino acid changes at at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402 and 413 according to EU numbering.

In another aspect, the invention provides polypeptides containing variant Fc regions with increased FcgammaRIIb binding activity and increased pI, containing amino acid changes according to any of the following options (1)-(16): (1) positions 234, 238, 250, 264, 307, 311, 330 and 343; (2) positions 234, 238, 250, 264, 307, 311, 330 and 413; (3) positions 234, 238, 250, 264, 267, 307, 311, 330 and 343; (4) positions 234, 238, 250, 264, 267, 307, 311, 330 and 413; (5) positions 234, 238, 250, 267, 307, 311, 330 and 343; (6) positions 234, 238, 250, 267, 307, 311, 330 and 413; (7) positions 234, 238, 250, 307, 311, 330 and 343; (8) positions 234, 238, 250, 307, 311, 330 and 413; (9) positions 238, 250, 264, 267, 307, 311, 330 and 343; (10) positions 238, 250, 264, 267, 307, 311, 330 and 413; (11) positions 238, 250, 264, 307, 311, 330 and 343; (12) positions 238, 250, 264, 307, 311, 330 and 413; (13) positions 238, 250, 267, 307, 311, 330 and 343; (14) positions 238, 250, 267, 307, 311, 330 and 413; (15) positions 238, 250, 307, 311, 330 and 343; and (16) positions 238, 250, 307, 311, 330 and 413; according to EU numbering.

In addition, amino acid changes for other purposes can be combined in the variant Fc region described herein. For example, amino acid substitutions can be added that improve FcRn binding activity (Hinton et al., J. Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33 ):23514-23524 (2006); Intl. Immunol. 18(12): 1759-1769 (2006); ; WO 2006/019447; WO 2006/053301; and WO 2009/086320), and amino acid substitutions to improve heterogeneity or stability of the antibody (WO 2009/041613). Alternatively, polypeptides with the property of promoting antigen clearance, which are described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides with the property of specifically binding to a target tissue, which are described in WO 2013/180200, polypeptides with the property of rebinding to multiple antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with the variant Fc region described herein. Alternatively, the amino acid changes described in EP1752471 and EP1772465 can be combined in the CH3 of the variant Fc region described herein to confer the ability to bind other antigens. Alternatively, for the purpose of prolonging plasma retention, amino acid changes that reduce the pI of the constant region (WO 2012/016227) can be combined in the variant Fc region described herein. Alternatively, to promote cellular uptake, amino acid changes that increase the pI of the constant region (WO 2014/145159) can be combined in the variant Fc region described herein. Alternatively, to promote elimination of a target molecule from plasma, amino acid changes that increase the pI of the constant region (WO 2016/125495) can be combined in the variant Fc region described herein. In one embodiment, such a change may include, for example, a substitution in at least one position selected from the group consisting of 311, 343, 384, 399, 400 and 413 in accordance with the EU numbering. In a further embodiment, such a substitution may be a Lys or Arg amino acid substitution at each position.

Amino acid changes to increase human FcRn binding activity at acidic pH can also be combined in the variant Fc region described herein. In particular, such changes may include, for example, the replacement of Leu with Met at position 428 and the replacement of Ser with Asn at position 434 in accordance with EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); replacement of Ala with Asn at position 434 (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); replacing Tyr with Met at position 252, replacing Thr with Ser at position 254, and replacing Glu with Thr at position 256 (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)); replacing Gin with Thr at position 250 and replacing Leu with Met at position 428 (Hinton et al., J. Immunol. 176(l):346-356 (2006)); replacement of His with Asn at position 434 (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011), and changes described in WO 2010/106180, WO 2010/045193, WO 2009/058492 , WO 2008/022152, WO 2006/050166, WO 2006/053301, WO 2006/031370, WO 2005/123780, WO 2005/047327, WO 2005/037867, WO 2004/035752 or W O 2002/060919. Such changes may include for example, at least one change selected from the group consisting of a change from Leu to Met at position 428, a change from Ala to Asn at position 434, and a change from Thr to Tyr at position 436. Such changes may further include a change from Arg to Gln at position 438 and/or replacing Glu with Ser at position 440 (WO 2016/125495).

Typical bispecific antibodies to CCL2

In one embodiment, the invention provides a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second distinct antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

A) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

B) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

B) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14;

or

D) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (6) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43;

and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

E) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

Ε) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 51; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

G) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

And) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30;

or

K) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 3; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain.

In one embodiment, the in vivo clearance rate (ml/day/kg) of human CCL2 following administration of a bispecific antibody comprising a wild-type human IgG1 isotype heavy chain constant domain (or Fc domain thereof) is at least twofold (in in one embodiment is at least 5 times greater, in one embodiment is at least 10 times greater, in one embodiment is at least 20 times greater) the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody containing the constant domain of the heavy chain of the human IgG1 isotype (or its Fc domain) with Fc-gamma receptor silencing, containing mutations L234A, L235A, P329G, when a pre-formed immune complex consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg human CCL2 were administered in a single dose of 10 ml/kg to FcRn transgenic mice.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35; and a VL domain containing the amino acid sequence of SEQ ID NO:40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain.

In one embodiment, the in vivo clearance rate of human CCL2 (ml/day/kg) following administration of a bispecific antibody comprising the wild-type human IgG1 isotype heavy chain constant domain (or Fc domain thereof) is at least 15-fold greater than in particular, exceeds at least 20 times the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody containing the heavy chain constant domain of the human IgG1 isotype (or its Fc domain) with Fc gamma receptor silencing, containing mutations L234A, L235A, P329G, when a preformed immune complex consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg of human CCL2 was administered in a single dose of 10 ml/kg to FcRn transgenic mice.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) a CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X1 is V, I or Η, X ² is Ρ or Η and X ³ is Η or G, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59;

And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain containing (d) CDR-L1 containing the amino acid sequence KAX1EDIYNRX2A from SEQ ID NO: 79, where X1 is F or T and X is R or L, (e) CDR-L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) a CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X1 is V, I or Η, X ² is Ρ or Η and X ³ is Η or G, (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59, (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF from SEQ ID NO: 63, (e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG from SEQ ID NO: 64, (e) FR-H3 containing the amino acid the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR from SEQ ID NO: 65, and (g) FR-H4 containing the amino acid sequence WGQGTLVTVSS from SEQ ID NO: 66;

And

a VL domain containing(s) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (j) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (k) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62, (l) FR-L1 containing the amino acid sequence EIVLTQSPATLSLSPGERATLSC from SEQ ID NO: 67, (n) FR-L2 containing the amino acid sequence WYQQKPGQAPRLLIY from SEQ ID NO: 68, (p) FR-L3 containing the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC from SEQ ID NO:69, and (p) FR-L4 containing amino acid sequence GQGTKVEIK from SEQ ID NO: 70; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D or E, (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E, (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS from SEQ ID NO: 82, ( e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG from SEQ ID NO: 83, (f) FR-H3 containing the amino acid sequence RVTITADTSSTAYMELSSLRSEDTAVYYCAR from SEQ ID NO: 84, and (g) FR-H4 containing the amino acid sequence WGQGTTVTVSS from SEQ ID NO:85;

And

a VL domain containing(s) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (k) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, (k) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R, (m) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC from SEQ ID NO: 86, (n) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIH from SEQ ID NO: 87, (p) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC from SEQ ID NO: 88, and (p) FR-L4 containing amino acid sequence FGGGTKVEIK from SEQ ID NO: 89.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

A) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

D) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

E) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

Ε) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

G) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

And) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

K) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

K) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

Μ) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

Η) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

Π) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

Ρ) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

C) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

Τ) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain. In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein

A) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X represents I, (b) CDR-H2 containing the amino acid sequence

GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ is V, X ² is P and X ³ is H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F and X ² is R, (e) CDR-L2 containing the amino acid the sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W; or

B) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, X ² represents P and X represents H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F and X ² is R, (e) CDR-L2 containing the amino acid the sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W; or

B) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R; or

D) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X3 represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

E) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X1 is F or T and X is R or L, (e) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R; or

E) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, Χ ² represents Ρ or Η and Χ ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

G) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

i) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

K) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

K) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

M) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

H) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

P) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or Τ, (6) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R;

or

Ρ) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R; or

C) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R; or

T) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or Η, X ² represents P or Η and X ³ represents Η or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (6) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain.

In one embodiment, a bispecific antibody described herein

i) blocks the binding of CCL2 to its receptor CCR2 in vitro (reporter gene assay, IC ₅₀ =0.5 nM); and/or

ii) inhibits CCL2-mediated chemotaxis of myeloid cells in vitro (IC ₅₀ =1.5 nM); and/or

iii) is cross-reactive with cynomolgus and human CCL2.

In one embodiment, the bispecific antibody described herein is not cross-reactive with other human CCL homologues, in particular, it exhibits 100-fold less binding to other CCL homologs (selected from the group of CCL8, CCL7 and CCL13) compared to binding to CCL2.

In one embodiment, a bispecific antibody described herein binds to the first and second epitopes on human CCL2 in an ion-dependent manner.

In one embodiment, a bispecific antibody described herein binds to human CCL2 in a pH-dependent manner, and wherein both the first antigen binding site and the second antigen binding site bind to CCL2 with greater affinity at neutral pH than at acidic pH.

In one embodiment, a bispecific antibody described herein binds to human CCL2 with 10 times greater affinity at pH 7.4 than at pH 5.8.

In one embodiment, a bispecific antibody described herein comprises two IgG1 heavy chain constant domains (or Fc domain thereof) containing (independent of or in addition to the mutations described above) the following mutations (Kabat EU numbering)

i) S354C and T366W in one of the heavy chain constant domains,

ii) Y349C, T366S, L368A, Y407V in another of the heavy chain constant domains.

In one embodiment, the bispecific antibody comprises an Fc domain of the human IgG1 isotype.

In one embodiment, the bispecific antibody comprises a human IgG1 isotype heavy chain constant domain. In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and/or A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iii) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering)

Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L234Y, P238D, T250V, V264I, T307P and/or A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) M428L, N434A and Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises a human IgG1 heavy chain constant domain (or Fc domain thereof) containing one or more of the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein

A) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

D) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 5; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

wherein said bispecific antibody is a full-length antibody of the human IgG isotype (preferably the human IgG1 isotype)c

a) a first light chain and a first heavy chain of a first antibody containing said first antigen-binding site of corresponding i); And

b) a second light chain and a second heavy chain of a second antibody containing said second antigen binding site according to the corresponding ii), and wherein the VL and VH variable domains in the second light chain and the second heavy chain of the second antibody are replaced with each other; And

wherein in the CL constant domain of the first light chain in a) the amino acid at position 124 is replaced by lysine (K) (numbering in accordance with Kabat) and the amino acid in position 123 is replaced by lysine (K) (numbering in accordance with Kabat),

and wherein in the CH1 constant domain of the first heavy chain in a) the amino acid at position 147 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat).

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein

i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO:93;

wherein said bispecific antibody is a (full-length) antibody with

a) a first light chain of kappa or lambda and a first heavy chain of the IgG1 isotype containing said first antigen-binding site of i); And

b) a second kappa or lambda light chain and a second heavy chain of IgG1 of the IgG1 isotype, containing said second antigen-binding site according to ii), and wherein the variable domains VL and VH in the second light chain and the second heavy chain of the second antibody are replaced by each other; And

wherein in the CL constant domain of the first light chain in a) the amino acid at position 124 is replaced by lysine (K) (numbering in accordance with Kabat) and the amino acid in position 123 is replaced by lysine (K) (numbering in accordance with Kabat),

and wherein in the CH1 constant domain of the first heavy chain in a) the amino acid at position 147 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat).

In one embodiment, such a bispecific antibody contains the following mutations (Kabat EU numbering)

i) S354C and T366W in one of the heavy chain constant domains,

ii) Y349C, T366S, L368A, Y407V in another of the heavy chain constant domains.

In one embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one alternative embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iii) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one alternative embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

In one alternative embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) M428L, N434A and Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one alternative embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 112, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 113, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 114, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 115 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 112, a polypeptide containing the amino acid sequence of SEQ ID NO: 113, a polypeptide containing the amino acid sequence of SEQ ID NO: 114, and a polypeptide containing the amino acid sequence of SEQ ID NO: 115.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 116, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 117, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 118, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 119 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 116, a polypeptide containing the amino acid sequence of SEQ ID NO: 117, a polypeptide containing the amino acid sequence of SEQ ID NO: 118, and a polypeptide containing the amino acid sequence of SEQ ID NO: 119.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 120, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 121, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 122, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 123 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 120, a polypeptide containing the amino acid sequence of SEQ ID NO: 121, a polypeptide containing the amino acid sequence of SEQ ID NO: 122, and a polypeptide containing the amino acid sequence of SEQ ID NO: 123.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 120, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 121, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 122, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 123 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 120, a polypeptide containing the amino acid sequence of SEQ ID NO: 121, a polypeptide containing the amino acid sequence of SEQ ID NO: 122, and a polypeptide containing the amino acid sequence of SEQ ID NO: 123.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 155, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 156, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 157, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 158 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 155, a polypeptide containing the amino acid sequence of SEQ ID NO: 156, a polypeptide containing the amino acid sequence of SEQ ID NO: 157, and a polypeptide containing the amino acid sequence of SEQ ID NO: 158.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 159, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 160, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 161, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 162 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 159, a polypeptide containing the amino acid sequence of SEQ ID NO: 160, a polypeptide containing the amino acid sequence of SEQ ID NO: 161, and a polypeptide containing the amino acid sequence of SEQ ID NO: 162.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 163, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 164, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 165, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 166 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 163, a polypeptide containing the amino acid sequence of SEQ ID NO: 164, a polypeptide containing the amino acid sequence of SEQ ID NO: 165, and a polypeptide containing the amino acid sequence of SEQ ID NO: 166.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 167, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 168, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 169, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 170 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 167, a polypeptide containing the amino acid sequence of SEQ ID NO: 168, a polypeptide containing the amino acid sequence of SEQ ID NO: 169, and a polypeptide containing the amino acid sequence of SEQ ID NO: 170.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 171, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 172, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 173, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 174 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 171, a polypeptide containing the amino acid sequence of SEQ ID NO: 172, a polypeptide containing the amino acid sequence of SEQ ID NO: 173, and a polypeptide containing the amino acid sequence of SEQ ID NO: 174.

In one embodiment of the invention, an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein

i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

wherein said bispecific antibody is a (full-length) human IgG1 isotype antibody with

a) a first light chain of kappa or lambda and a first heavy chain of the IgG1 isotype containing said first antigen-binding site of i); And

b) a second kappa or lambda light chain and a second heavy chain of IgG1 of the IgG1 isotype, containing said second antigen-binding site according to ii), and wherein the variable domains VL and VH in the second light chain and the second heavy chain of the second antibody are replaced by each other; And

wherein in the CL constant domain of the first light chain in a) the amino acid at position 124 is replaced by lysine (K) (numbering in accordance with Kabat) and the amino acid in position 123 is replaced by lysine (K) (numbering in accordance with Kabat),

and wherein in the CH1 constant domain of the first heavy chain in a) the amino acid at position 147 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat).

In one embodiment, such a bispecific antibody contains the following mutations (Kabat EU numbering)

i) S354C and T366W in one of the heavy chain constant domains,

ii) Y349C, T366S, L368A, Y407V in another of the heavy chain constant domains.

In one embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iii) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one embodiment, a bispecific antibody described herein comprises an IgG1 heavy chain constant domain (or Fc domain thereof) containing the following mutations (Kabat EU numbering):

Q311R and P343R (suitable for increasing pI to enhance antigen uptake).

In one alternative embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) M428L, N434A and Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In one alternative embodiment, such a bispecific antibody further contains the following mutations (Kabat EU numbering):

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 124, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 125, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 126, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 127 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 124, a polypeptide containing the amino acid sequence of SEQ ID NO: 125, a polypeptide containing the amino acid sequence of SEQ ID NO: 126, and a polypeptide containing the amino acid sequence of SEQ ID NO: 127.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 128, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 129, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 130, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 131 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 128, a polypeptide containing the amino acid sequence of SEQ ID NO: 129, a polypeptide containing the amino acid sequence of SEQ ID NO: 130, and a polypeptide containing the amino acid sequence of SEQ ID NO: 131.

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 132, a polypeptide containing an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 133, a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 134, and a polypeptide comprising an amino acid sequence that is at least 98% or 99% identical to the sequence of SEQ ID NO: 135 .

In a specific embodiment, the invention provides an (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2, wherein the bispecific antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO: 132, a polypeptide containing the amino acid sequence of SEQ ID NO: 133, a polypeptide containing the amino acid sequence of SEQ ID NO: 134, and a polypeptide containing the amino acid sequence of SEQ ID NO: 135.

Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CCL2 antibody (bispecific or monospecific) described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising one or all of the VLs and/or an amino acid sequence comprising one or all of the VHs of a mono- or bispecific antibody (eg, the light and/or heavy chains of an antibody). In a further embodiment, one or more vectors (eg, expression vectors) containing such a nucleic acid are provided. In a further embodiment, a host cell containing such a nucleic acid is provided. In one such embodiment, the host cell contains (for example, has been transformed): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing a VL antibody and an amino acid sequence containing a VH antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing a VL antibody, and a second vector containing a nucleic acid that encodes an amino acid sequence containing a VH antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, a HEK293 cell, or a lymphoid cell (eg, a Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-CCL2 antibody is provided, comprising culturing a host cell containing a nucleic acid encoding the antibody provided above under conditions suitable for expression of the antibody, and optionally isolating the antibody from the host cell (or cell culture medium). owner).

To recombinantly produce anti-CCL2 cells, such nucleic acid can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5648237, US 5789199 and US 5840523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) Once expressed, the antibody can be isolated from the bacterial cell paste into a soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for the cloning or expression of antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in an antibody with a partially or fully human glycosylation profile . See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, N. et al., Nat. Biotech. 24 (2006) 210-215.

Host cells suitable for expressing a glycosylated antibody are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7); a human embryonic kidney cell line (293 or 293 cells, described, for example, in Graham F.L. et al., J. Gen Virol. 36 (1977) 59-74); newborn hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, described, for example, in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK; gray rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, described, for example, in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68; other suitable mammalian cell lines include Chinese hamster ovary (CHO) cells (Urlaub G). . et al., Proc. Natl. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2/0. , see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

In another aspect, the invention is based in part on the discovery that the modified monospecific antibodies described herein exhibit improved pH-dependent binding properties and are therefore particularly useful for generating the bispecific antibodies of the invention.

Monospecific antibodies to CCL2 with pH-dependent binding properties

In one embodiment, the invention provides an (isolated) (monospecific) antibody that (specifically) binds to human CCL2,

wherein the antibody contains

A) a VH domain containing (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, wherein X ¹ is V, I or H, X ² is P or H and X ³ is H or G, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62;

or

B) a VH domain containing (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X represents D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E;

And

a VL domain containing (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

In one embodiment, the invention provides an (isolated) (monospecific) antibody that (specifically) binds to human CCL2,

wherein the antibody contains

A) a VH domain containing the amino acid sequence of SEQ ID NO: 71;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75; or

B) VH domain containing the amino acid sequence SEQ ID NO: 72;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75; or

B) a VH domain containing the amino acid sequence of SEQ ID NO: 73;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75; or

D) VH domain containing the amino acid sequence SEQ ID NO: 74;

and a VL domain containing the amino acid sequence of SEQ ID NO: 75; or

E) VH domain containing the amino acid sequence SEQ ID NO: 90;

and a VL domain containing the amino acid sequence of SEQ ID NO: 93; or

E) a VH domain containing the amino acid sequence of SEQ ID NO: 91;

and a VL domain containing the amino acid sequence of SEQ ID NO: 93; or

G) VH domain containing the amino acid sequence SEQ ID NO: 92;

and a VL domain containing the amino acid sequence of SEQ ID NO: 93; or

I) VH domain containing the amino acid sequence SEQ ID NO: 90;

and a VL domain containing the amino acid sequence of SEQ ID NO: 94.

Methods and compositions for diagnosis and detection

In some embodiments, any of the anti-CCL2 mono- or bispecific antibodies provided herein are useful for detecting the presence of CCL2 in a biological sample. In the context of this document, the term “identification” includes quantitative or qualitative identification. In certain embodiments, the biological sample comprises a cell or tissue, such as an immune cell, or T cell infiltrates, and/or tumor cells.

In one embodiment, a mono- or bispecific anti-CCL2 antibody is provided for use in a diagnostic or detection method. In an additional aspect, a method is provided for detecting the presence of CCL2 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with a mono- or bispecific anti-CCL2 antibody described herein under conditions that allow the mono- or bispecific anti-CCL2 antibody to bind to CCL2, and determining whether a complex has formed between the mono- or bispecific anti-CCL2 antibodies. or a bispecific antibody to CCL2 and CCL2. Such a method may be an in vitro or in vivo method. In one embodiment, a mono- or bispecific anti-CCL2 antibody is used to select subjects suitable for therapy with a mono- or bispecific anti-CCL2 antibody, such as when CCL2 is a biomarker for patient selection.

In some embodiments, the invention provides labeled mono- or bispecific antibodies to CCL2. Labels include, but are not limited to, directly detectable tags or moieties (e.g., fluorescent, chromophore, electron dense, chemiluminescent, and radioactive tags), as well as indirectly detectable moieties, such as enzymes or ligands, such as through an enzymatic reaction or molecular interaction. Typical labels include, but are not limited to, the radioactive isotopes ^32P , ^14C , ^125I , ^3H and ^131I , fluorophores such as rare earth metal chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g. firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin tags, bacteriophage tags, stable free radicals, etc.

D. Pharmaceutical compounds

Pharmaceutical formulations of the mono- or bispecific anti-CCL2 antibody described herein are prepared by mixing such antibody, having the required purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980 )) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (containing less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes) and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include agents for dispersing drugs in the interstitial space, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g., soluble human hyaluronidase glycoprotein PH-20, such as rhuPH20 ( ^HYLENEX® , Baxter International, Inc.). Some exemplary sHASEGPs and their uses, including rhuPH20, are described in US Patent Publications Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter formulations containing a histidine acetate buffer.

The composition provided herein may also contain more than one active ingredient if necessary for the particular indication being treated, preferably those that have complementary activities and do not adversely affect each other. For example, it may be desirable to further provide. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

Active ingredients can also be encapsulated in microcapsules obtained, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

Slow-release formulations can be obtained. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of molded articles, such as films or microcapsules.

Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

E. Therapeutic Methods and Compositions

Any of the mono- or bispecific antibodies to CCL2 provided herein can be used in therapeutic methods.

In one aspect, a mono- or bispecific anti-CCL2 antibody is provided for use as a drug. In additional aspects, a mono- or bispecific anti-CCL2 antibody is provided for use in the treatment of cancer. In certain embodiments, a mono- or bispecific anti-CCL2 antibody is provided for use in a method of treatment. In certain embodiments, the invention provides a mono- or bispecific anti-CCL2 antibody for use in a method of treating an individual having cancer, comprising administering to the individual an effective amount of a mono- or bispecific anti-CCL2 antibody.

In additional embodiments, the invention provides a mono- or bispecific anti-CCL2 antibody that inhibits immunosuppression in tumors and thereby renders the tumor more susceptible to immunostimulatory agents such as anti-PD1, anti-PDL-1 antagonists, and the like.

Therefore, one aspect of the invention provides a combination of mono- or bispecific anti-CCL2 antibodies described herein with cancer immunotherapies, such as anti-PD1, anti-PDL-1 antagonists, and the like.

As used herein, the term “cancer” may be, for example, lung cancer, non-small cell lung cancer (NSCLC), bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal cell carcinoma pelvis, mesothelioma, hepatic cell carcinoma, gallbladder cancer, central nervous system (CNS) neoplasms, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers or a combination of one or more of the above cancers.

The "individual" in accordance with any of the above embodiments is preferably a human. In an additional aspect, the invention provides the use of a mono- or bispecific anti-CCL2 antibody in the manufacture or production of a drug. In one embodiment, the drug is for the treatment of cancer. In a further embodiment, the drug is for use in a method of treating cancer, comprising administering to an individual having cancer an effective amount of the drug. In a further embodiment, the drug is intended to induce cell-mediated lysis of cancer cells. In a further embodiment, the drug is for use in a method of inducing cell-mediated lysis of cancer cells in a subject having cancer, comprising administering to the subject an amount of the drug effective to induce apoptosis and/or inhibit proliferation of cancer cells. The "individual" in accordance with any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating cancer. In one embodiment, the method includes administering to an individual having cancer an effective amount of a mono- or bispecific anti-CCL2 antibody. The "individual" in accordance with any of the above embodiments may be a human.

In a further aspect, the invention provides a method for inducing cell-mediated lysis of cancer cells in an individual having cancer. In one embodiment, the method includes administering to an individual an effective amount of a mono- or bispecific anti-CCL2 antibody to induce cell-mediated lysis of cancer cells in the individual having cancer. In one embodiment, the “individual” is a human.

In an additional aspect of the invention, a mono- or bispecific antibody to CCL2 is provided for use in the treatment of inflammatory diseases or autoimmune diseases. In certain embodiments, the invention provides a mono- or bispecific anti-CCL2 antibody for use in a method of treating an individual having an inflammatory disease or an autoimmune disease, comprising administering to the individual an effective amount of a mono- or bispecific anti-CCL2 antibody.

In a further aspect, the present invention provides pharmaceutical compositions containing any of the anti-CCL2 mono- or bispecific antibodies provided herein, for example, for use in any of the above therapeutic modalities. In one embodiment, the pharmaceutical composition comprises any of the anti-CCL2 mono- or bispecific antibodies provided herein and a pharmaceutically acceptable carrier.

In some embodiments, the inflammatory disease or autoimmune disease is an autoimmune disorder, an inflammatory disorder, a fibrotic disorder, a granulocytic (neutrophil or eosinophilic) disorder, a monocytic disorder, or a lymphocytic disorder, or a disorder associated with an increase in the number or distribution of normal or aberrant tissue resident cells ( such as mast cells, macrophages or lymphocytes) or stromal cells (such as fibroblasts, myofibroblasts, smooth muscle cells, epithelial or endothelial cells). In some embodiments, the disorder is a pulmonary disorder. In some embodiments, the pulmonary disorder is associated with granulocytic (neutrophilic and/or eosinophilic) pulmonary inflammation, infection-induced pulmonary conditions (including those associated with viral (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus), bacterial or fungal (eg, Aspergillus) triggers. In some embodiments, the disorder is an allergen-induced pulmonary condition, an environmental toxicant-induced pulmonary condition (eg, asbestosis, silicosis, or beryllium disease), a gastric aspiration-induced pulmonary condition, or an associated pulmonary condition. an immune dysregulation disorder; or an inflammatory condition with a genetic predisposition, such as cystic fibrosis. In some embodiments, the disorder is a physical trauma-induced pulmonary condition (eg, ventilatory injury), emphysema, smoking-induced emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangiomyomatosis, acute. lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (eg, community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, and severe pneumonia), exacerbation of respiratory diseases, and acute respiratory distress syndrome (ARDS)). In some embodiments, the inflammatory pulmonary disorder is COPD.

In some embodiments, the inflammatory pulmonary disorder is asthma. In some embodiments, asthma is persistent chronic severe asthma with acute events of worsening symptoms (exacerbations or flares) that can be life-threatening. In some embodiments, the asthma is atopic (also known as allergic) asthma, non-allergic asthma (e.g., often caused by infection with a respiratory virus (e.g., influenza virus, parainfluenza virus, rhinovirus, human metapneumovirus, and respiratory syncytial virus), or inhalation of an irritant (air pollutants, smog, fuel particles, volatile chemicals and gases indoors or outdoors, or even cold dry air).

In some embodiments, asthma is intermittent or exercise-induced asthma due to acute or chronic first-hand or second-hand smoke (typically cigarettes, cigars, cigarettes), inhalation or "vaping" (of tobacco, marijuana, or other similar substances), or asthma caused by recent use of aspirin or similar NSAIDs. In some embodiments, asthma is mild or corticosteroid-induced asthma, newly diagnosed and untreated asthma, or not previously requiring chronic use of inhaled topical or systemic steroids to control symptoms (cough, wheezing, shortness of breath/shortness of breath, or chest pain ). In some embodiments, the asthma is chronic, corticosteroid-resistant asthma, corticosteroid-refractory asthma, uncontrolled asthma while taking corticosteroids or other medications to control chronic asthma. In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, neutrophilic disorder, or eosinophilic disorder is pulmonary fibrosis. In some embodiments, the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder is esophagitis, allergic rhinitis, non-allergic rhinitis, rhinosinusitis with polyps, nasal polyposis, bronchitis, chronic pneumonia, allergic bronchopulmonary aspergillosis, respiratory tract inflammation, allergic rhinitis, bronchiectasis and/or chronic bronchitis.

In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophil or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder is arthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is osteoarthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, early arthritis, rheumatoid polyarthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, psoriatic arthritis, and/or arthritis due to injury.

In some embodiments, the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder is a gastrointestinal inflammatory disease. In some embodiments, the gastrointestinal inflammatory disease is IBD (inflammatory bowel disease), ulcerative colitis (UC), Crohn's disease (CD), colitis (e.g., environmentally induced colitis (e.g., caused by or associated with a therapeutic regimen, such as chemotherapy, radiation therapy, etc.), infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis due to conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, gastroenteritis, infectious gastritis or enterocolitis (eg, chronic active gastritis with Helicobacter pylori infection) and other forms of gastrointestinal inflammation caused by an infectious agent, or unspecified colitis.

In some embodiments, an autoimmune disorder, an inflammatory disorder, a fibrotic disorder, a granulocytic (neutrophil or eosinophilic) disorder, a monocytic disorder, or a lymphocytic disorder, or a lymphocytic disorder, or a disorder associated with an increase in the number or distribution of normal or aberrant tissue resident cells (such as mast cells, macrophages, or lymphocytes) or stromal cells (such as fibroblasts, myofibroblasts, smooth muscle cells, epithelial or endothelial cells) is lupus, systemic lupus erythematosus (SLE), or one or more organ-specific manifestations of lupus (eg, lupus nephritis (LN), affecting the kidneys, or extrarenal lupus (ERL), affecting hematopoietic and/or lymphoid organs (lymph nodes, spleen, thymus and associated lymphatic vessels), and/or joints, and/or other organs, but not necessarily the kidneys). In some embodiments, the autoimmune disorder, inflammatory disorder, or fibrotic disorder is associated with sepsis and/or trauma, HIV infection, or idiopathic (of unknown etiology), such as ANCA-associated vasculitis (AAV), granulomatosis with angiopathy (formerly known as Wegener's granulomatosis), disease Behçet, cardiovascular disease, eosinophilic bronchitis, Reiter's syndrome, SEA syndrome (seronegativity, enthesopathy, arthropathy syndrome), ankylosing spondylitis, dermatomyositis, scleroderma, eg, systemic scleroderma, also called systemic sclerosis, vasculitis (eg, giant cell arteritis (GCA) , also called temporal arteritis, cranial arteritis or Horton's disease), myositis, polymyositis, dermatomyositis, polyarteritis nodosa, arteritis, polymyalgia rheumatica, sarcoidosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis , pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, pemphigus, e.g. pemphigus vulgaris, atherosclerosis, lupus, Still's syndrome, myasthenia gravis, celiac disease, multiple sclerosis (MS), relapsing-remitting (RRMS), primary progressive (PPMS) or secondary progressive (SPMS) subtypes, Guillain-Barré disease, type 1 diabetes mellitus (T1DM), insulin-independent diabetes mellitus (NIDDM) or juvenile type, thyroiditis (for example, Graves' disease), celiac disease, Churg-Strauss syndrome, muscle pain, hypereosinophilic syndrome, edematous reactions, including episodic angioedema, helminthic infections, onchocercal dermatitis, eosinophilic esophagitis, eosinophilic enteritis, eosinophilic colitis, obstructive sleep apnea syndrome, endomyocardial fibrosis, Addison's disease, Raynaud's disease or phenomenon, autoimmune hepatitis oh, illness graft-versus-host disease (GvHD) or organ transplant rejection.

The antibody of the invention (and any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal, and, if necessary for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be administered in any convenient manner, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or continuous. Various dosing schedules are provided herein, including, but not limited to, single or multiple dosing at different times, bolus dosing, and pulse infusion.

The antibodies of the invention are formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to the practitioner. The antibody is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. Other agents are generally used in the same dosages and administered by the routes described herein, or in dosages comprising from about 1 to 99% of the dosages described herein, or in any dosage and by any route that are empirically/clinically determined to be appropriate .

For the prevention or treatment of a disease, the appropriate dose of an antibody of the invention (used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically. purpose, previous therapy, the patient's medical history and response to the antibody, and the treating physician's judgment. The antibody is preferably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, the initial candidate dosage of antibody to be administered to a patient may range from about 1 μg/kg to 15 mg/kg (e.g., 0.5 mg/kg 10 mg/kg), for example, in one or more separate injections or by continuous infusion. One typical daily dose can range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. In the case of repeated administrations over several days or more, depending on the condition, treatment is generally continued until the required degree of suppression of the symptoms of the disease is achieved. One typical antibody dosage ranges from about 0.05 mg/kg to about 10 mg/kg. Thus, the patient may be administered one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof). Such doses may be administered at intervals, for example, once a week or every three weeks (eg, so that the patient receives from about two to about twenty or, for example, about six doses of the antibody). An initial higher loading dose may be administered followed by one or more lower doses. A typical dosing schedule includes an initial loading dose of approximately 4 mg/kg followed by a weekly maintenance dose of approximately 2 mg/kg of antibody. In this case, other dosage schedules can be used. The effectiveness of such therapy can be easily monitored using traditional methods and tests.

II. Finished goods

In another aspect of the invention, a finished product is provided containing materials useful in the treatment, prevention and/or diagnosis of the above-described disorders. The finished product contains a container and a label or package insert on or associated with it. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. Containers can be made from a number of materials, such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective for the treatment, prevention and/or diagnosis of a pathological condition, and may have a sterile inlet (for example, the container may be an intravenous solution bag or a vial with a stopper pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody according to the invention. The label or package insert states that the composition is used to treat the selected pathological condition. In addition, the finished product may comprise (a) a first container containing a composition therein, said composition comprising an antibody of the invention; and (b) a second container containing a composition therein, said composition containing an additional cytotoxic or other therapeutic agent. The finished product in this embodiment may further include a package insert indicating that the compositions can be used to treat a particular pathological condition. Alternatively or additionally, the finished product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also contain other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

The following are specific embodiments of the invention:

1. A bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second distinct antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein the bispecific antibody comprises an isotype Fc domain Human IgG, preferably of the IgG1 isotype.

2. The bispecific antibody according to embodiment 1, wherein

A) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

B) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35;

and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19;

and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

B) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14;

or

D) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

E) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

E) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 51; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 43; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

G) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

And) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30;

or

K) i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and (c) a CDR -H3 containing the amino acid sequence SEQ ID NO: 3; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30.

3. Bispecific antibody according to embodiment 2,

and

A) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid the sequence SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing the amino acid sequence of SEQ ID NO: 40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 47, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

B) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) CDR-H2 containing the amino acid the sequence SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing the amino acid sequence of SEQ ID NO: 40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 23, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and a VL domain containing the amino acid sequence of SEQ ID NO: 24, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

B) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, (b) a CDR-H2 containing the amino acid the sequence SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 35; and a VL domain containing the amino acid sequence of SEQ ID NO: 40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 15, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 11; and a VL domain containing the amino acid sequence of SEQ ID NO: 16, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14;

or

D) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 23, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 containing the amino acid the sequence SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 19; and a VL domain containing the amino acid sequence of SEQ ID NO: 24, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 47, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

E) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 31, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 containing the amino acid the sequence SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 27; and a VL domain containing the amino acid sequence of SEQ ID NO: 32, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 47, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

E) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 55, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, (b) a CDR-H2 containing the amino acid the sequence SEQ ID NO: 50, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 51; and a VL domain containing the amino acid sequence of SEQ ID NO: 56, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 52; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 53, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 54; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 47, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46;

or

G) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 15, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 containing the amino acid the sequence SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing the amino acid sequence of SEQ ID NO: 16, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 23, the VH domain comprising (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and a VL domain containing the amino acid sequence of SEQ ID NO: 24, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 22;

or

And) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 15, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 9, (b) a CDR-H2 containing the amino acid the sequence SEQ ID NO: 10, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 11; and a VL domain containing the amino acid sequence of SEQ ID NO: 16, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 12; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 13, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 14; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 31, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; and a VL domain containing the amino acid sequence of SEQ ID NO: 32, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30;

or

K) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 7, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 containing the amino acid the sequence SEQ ID NO: 2, and (c) CDR-H3 containing the amino acid sequence SEQ ID NO: 3; and a VL domain containing the amino acid sequence of SEQ ID NO: 8, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 31, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, (b) a CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; and a VL domain containing the amino acid sequence of SEQ ID NO: 32, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 30.

4. Bispecific antibody according to any one of paragraphs. 1-3, wherein the bispecific antibody containing the Fc domain of the human IgG isotype is a bispecific antibody containing the heavy chain constant domain of the human IgGl isotype.

5. The bispecific antibody of Embodiment 4, wherein the in vivo clearance rate of human CCL2 (ml/day/kg) following administration of the bispecific antibody comprising the heavy chain constant domain of the wild-type human IgG1 isotype is at least twice as high (one-fold). in one embodiment, at least 5-fold, in one embodiment, at least 10-fold, in one embodiment at least 20-fold) the in vivo clearance rate for human CCL2 (ml/day/kg) after administration bispecific antibody containing the constant domain of the heavy chain of the human IgGl isotype with Fc-gamma receptor silencing, containing mutations L234A, L235A, P329G (EU numbering according to Kabat), when a preformed immune complex consisting of 20 mg/kg of each bispecific antibody and 0 .1 mg/kg human CCL2 was administered in a single dose of 10 ml/kg to FcRn transgenic mice.

6. An (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2 and a second antigen binding site that (specifically) binds to a second distinct epitope on human CCL2, wherein the bispecific antibody comprises an Fc domain human IgG isotype and moreover

i) said first antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 39, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 33 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 35;

and a VL domain containing the amino acid sequence of SEQ ID NO: 40, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 36; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and (e) CDR-L3 containing the amino acid sequence of SEQ ID NO: 38; And

ii) said second antigen binding site binds to the same epitope on CCL2 as an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 47, wherein the VH domain contains (a) a CDR-H1 containing the amino acid sequence of SEQ ID NO: 41 , (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 42, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 43; and a VL domain containing the amino acid sequence of SEQ ID NO: 48, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 44; (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 45, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 46.

7. The bispecific antibody of embodiment 6, wherein the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of the bispecific antibody comprising the heavy chain constant domain of the wild-type human IgG1 isotype is at least 15 times greater, in particular is at least 20 times the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody containing the heavy chain constant domain of the Fc gamma receptor silencing human IgG1 isotype containing the L234A, L235A, P329G mutations ( Kabat EU numbering) when a preformed immune complex consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg human CCL2 was administered in a single dose of 10 ml/kg to FcRn transgenic mice.

8. An (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) a CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ is V, I or H, X ² is P or H and X ³ is H or G, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; And

a VL domain containing (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

9. An (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

i) said first antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I or T, (b) a CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ is V, I or H, X ² is P or H and X ³ is H or G, (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59, (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTF from SEQ ID NO: 63, (e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG from SEQ ID NO: 64, (f) FR-H3 containing the amino acid sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR from SEQ ID NO: 65, and (g) FR-H4 containing the amino acid sequence WGQGTLVTVSS from SEQ ID NO: 66;

And

a VL domain containing(s) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (j) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (k) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62, (l) FR-L1 containing the amino acid sequence EIVLTQSPATLSLSPGERATLSC from SEQ ID NO: 67, (n) FR-L2 containing the amino acid sequence WYQQKPGQAPRLLIY from SEQ ID NO: 68, (p) FR-L3 containing the amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC from SEQ ID NO: 69, and (p) FR-L4 containing amino acid sequence GQGTKVEIK from SEQ ID NO: 70;

And

ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D or E, (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E, (d) FR-H1 containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGLTIS from SEQ ID NO: 82, ( e) FR-H2 containing the amino acid sequence WVRQAPGQGLEWMG from SEQ ID NO: 83, (f) FR-H3 containing the amino acid sequence RVTITADTSSTAYMELSSLRSEDTAVYYCAR from SEQ ID NO: 84, and (g) FR-H4 containing the amino acid sequence WGQGTTVTVSS from SEQ ID NO: 85;

And

a VL domain containing(s) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (j) CDR-L2, containing the amino acid sequence GATSLEH from SEQ ID NO: 80, (l) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R, (l) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC from SEQ ID NO: 86, (n) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIH from SEQ ID NO: 87, (p) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC from SEQ ID NO: 88, and (p) FR-L4 containing amino acid sequence FGGGTKVEIK from SEQ ID NO: 89.

10. An (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

A) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site contains a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

B) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

D) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

E) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

E) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

G) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

And) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 73; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

K) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

K) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

M) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 72; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

H) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

P) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 90; and a VL domain containing the amino acid sequence of SEQ ID NO: 94;

or

P) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

C) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 74; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93;

or

T) i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And

ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 92; and a VL domain containing the amino acid sequence of SEQ ID NO: 93.

11. An (isolated) bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2, and a second antigen binding site that (specifically) binds to a second epitope on human CCL2,

and

A) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, X ² represents P and X ³ represents H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F and X ² is R, (e) CDR-L2 containing the amino acid the sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W;

or

B) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, X ² represents P and X ³ represents H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (6) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F and X ² is R, (e) CDR-L2 containing the amino acid the sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W;

or

B) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO: 90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

D) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

E) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

E) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

G) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

i) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 73, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

K) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

K) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

M) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 72, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

H) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X represents W or R;

or

P) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ¹ represents P or H and X ² represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:90, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 94, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

P) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

C) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 74, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:91, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ₂ A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R;

or

T) i) said first antigen binding site contains a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity sequences with the amino acid sequence of SEQ ID NO: 71, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, where X ¹ represents V, I or H, X ² represents P or H and X ³ represents H or G, and (c) CDR-H3 containing amino acid sequence YDAHYGELDF from SEQ ID NO: 59; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And

ii) said second antigen binding site comprises a VH domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with amino acid sequence of SEQ ID NO:92, wherein the VH domain contains (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X is D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO:78, where X is D or E; and a VL domain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 93, wherein the VL domain contains (d) CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X ² is R or L, (e) CDR- L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W or R.

12. The bispecific antibody of any one of embodiments 8-11, wherein the bispecific antibody comprises an Fc domain of a human IgG isotype, preferably a human IgG1 isotype.

13. The bispecific antibody of any one of embodiments 8-11, wherein the bispecific antibody comprises a constant domain of a human IgG isotype, preferably a human IgG1 isotype.

14. The bispecific antibody according to any of the previous embodiments, wherein the bispecific antibody

i) blocks the binding of CCL2 to its receptor CCR2 in vitro (reporter gene assay, IC ₅₀ =0.5 nM); and/or

ii) inhibits CCL2-mediated chemotaxis of myeloid cells in vitro (IC ₅₀ =1.5 nM); and/or

iii) is cross-reactive with cynomolgus and human CCL2.

15. The bispecific antibody of any one of the preceding embodiments, wherein the bispecific antibody is not cross-reactive with other CCL homologues (shows 100-fold less binding to other CCL homologs (selected from the group of CCL8, CCL7 and CCL13) compared to binding to CCL2).

16. The bispecific antibody of any one of the preceding embodiments, wherein the bispecific antibody binds to the first and second epitopes on human CCL2 in an ion-dependent manner.

17. The bispecific antibody of any one of the preceding embodiments, wherein the bispecific antibody binds to human CCL2 in a pH-dependent manner and wherein both the first antigen binding site and the second antigen binding site bind to CCL2 with greater affinity at neutral pH than at acidic pH.

18. The bispecific antibody of any one of the preceding embodiments, wherein the bispecific antibody binds to human CCL2 with 10 times greater affinity at pH 7.4 than at pH 5.8.

19. A bispecific antibody according to any of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing one or more of the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (suitable for increasing pi to increase antigen uptake); and/or

ii) L234Y, L235W, G236N, P238D, T250V, V264I, T307PH268D, Q295L, K326T and/or A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

20. A bispecific antibody according to any of the preceding embodiments, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing one or more of the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L235W, G236N, H268D, Q295L, K326T and/or ADZOK (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

21. A bispecific antibody according to any of the preceding embodiments, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

22. A bispecific antibody according to any of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing one or more of the following mutations (Kabat EU numbering)

i) Q311R and/or P343R (suitable for increasing pI to enhance antigen uptake); and/or

ii) L234Y, P238D, T250V, V264I, T307V and/or A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); and/or

iii) M428L, N434A and/or Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); and/or

iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).

23. A bispecific antibody according to any of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing one or more of the following mutations (Kabat EU numbering)

i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) M428L, N434A and Y436T (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

24. A bispecific antibody according to any of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing one or more of the following mutations (Kabat EU numbering)

i) Q311R and P343R (suitable for increasing pI to enhance antigen uptake); And

ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs); And

iii) N434A (suitable for increasing affinity for FcRn to prolong plasma half-life); And

iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).

25. The bispecific antibody of any one of the preceding embodiments, wherein the bispecific antibody comprises two human IgG1 heavy chain constant domains containing the following mutations (EU numbering)

i) S354C and T366W in one of the heavy chain constant domains,

ii) Y349C, T366S, L368A, Y407V in another of the heavy chain constant domains.

26. An (isolated) antibody that (specifically) binds to human CCL2,

wherein the antibody contains

A) a VH domain containing (a) CDR-H1 containing the amino acid sequence SHYGXS from SEQ ID NO: 57, where X is I or T, (b) CDR-H2 containing the amino acid sequence GX ¹ IX ² IFX ³ TANYAQKFQG from SEQ ID NO: 58, wherein X ¹ is V, I or H, X ² is P or H and X ³ is H or G, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF of SEQ ID NO: 59;

And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60; (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62;

or

B) a VH domain containing (a) CDR-H1 containing the amino acid sequence HTYMH from SEQ ID NO: 76, (b) CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG from SEQ ID NO: 77, where X represents D or E, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH of SEQ ID NO: 78, where X is D or E;

And

a VL domain containing (d) a CDR-L1 containing the amino acid sequence KAX ¹ EDIYNRX ² A from SEQ ID NO: 79, where X ¹ is F or T and X is R or L, (e) a CDR-L2 containing the amino acid sequence GATSLEH of SEQ ID NO: 80, and (e) CDR-L3 containing the amino acid sequence QQFXSAPYT of SEQ ID NO: 81, where X is W or R.

27. An (isolated) antibody that (specifically) binds to human CCL2,

wherein the antibody contains

A) a VH domain containing the amino acid sequence of SEQ ID NO:71;

and a VL domain containing the amino acid sequence of SEQ ID NO:75;

or

B) VH domain containing the amino acid sequence of SEQ ID NO:72;

and a VL domain containing the amino acid sequence of SEQ ID NO:75;

or

B) a VH domain containing the amino acid sequence of SEQ ID NO:73;

and a VL domain containing the amino acid sequence of SEQ ID NO:75;

or

D) a VH domain containing the amino acid sequence of SEQ ID NO:74;

and a VL domain containing the amino acid sequence of SEQ ID NO:75;

or

E) a VH domain containing the amino acid sequence of SEQ ID NO:90;

and a VL domain containing the amino acid sequence of SEQ ID NO:93;

or

E) a VH domain containing the amino acid sequence of SEQ ID NO:91;

and a VL domain containing the amino acid sequence of SEQ ID NO:93;

or

G) VH domain containing the amino acid sequence of SEQ ID NO:92;

and a VL domain containing the amino acid sequence of SEQ ID NO:93;

or

I) a VH domain containing the amino acid sequence of SEQ ID NO:90;

and a VL domain containing the amino acid sequence of SEQ ID NO:94.

28. An isolated nucleic acid encoding an antibody according to any of the previous embodiments.

29. A host cell containing the nucleic acid of embodiment 28.

30. A method for producing an antibody, comprising culturing a host cell according to embodiment 29 so as to produce an antibody.

31. The method of embodiment 30, further comprising recovering the antibody from the host cell.

32. A pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1-25 and a pharmaceutically acceptable carrier.

33. The bispecific antibody according to any one of embodiments 1-25 for use as a drug.

34. The bispecific antibody of any one of embodiments 1-25 for use in the treatment of cancer.

35. The bispecific antibody of any one of embodiments 1-25 for use in the treatment of an inflammatory or autoimmune disease.

36. Use of the bispecific antibody according to any one of embodiments 1-25 in the production of a medicinal product.

37. Use according to embodiment 36, wherein the medicinal product is for the treatment of cancer.

38. Use according to embodiment 36, wherein the medicament is for the treatment of an inflammatory or autoimmune disease.

39. A method of treating an individual having cancer, comprising administering to the individual an effective amount of a bispecific antibody according to any one of claims. 1-25.

40. A method of treating an individual having an inflammatory or autoimmune disease, comprising administering to the individual an effective amount of a bispecific antibody according to any one of claims. 1-25.

The following examples and figures are provided to assist in understanding the present invention, the true scope of which is defined by the appended claims. It should be understood that modifications can be made to the set forth procedures without departing from the spirit of the invention.

Description of amino acid sequences

Anti-CCL2 antigen-binding fragments (variable regions and hypervariable regions (CDRs)) that bind to different epitopes:

Anti-CCL2 antigen-binding fragments with modified CDRs (variable regions and hypervariable regions (CDRs)):

Modified CNTQ888

Modified humanized 11K2

Typical light chain constant regions:

SEQ ID NO: 95 Typical Human Kappa Light Chain Constant Region

SEQ ID NO: 96 Typical Human Lambda Light Chain Constant Region

Typical heavy chain constant regions:

SEQ ID NO: 97 Typical human heavy chain constant region derived from IgG1

SEQ ID NO: 98 Generic human heavy chain constant region derived from IgG1 with mutations L234A, L235A and P329G (Fc gamma receptor silencing)

SEQ ID NO: 99 exemplary human heavy chain constant region derived from IgG1 (SG1-IgG1 allotype) SEQ ID NO: 100 exemplary human heavy chain constant region derived from IgG1 with mutations (allotype SG105-IgG1 Fc gamma receptor silencing )

SEQ ID NO: 101 SG1095 - Generic human heavy chain constant region derived from IgG1 containing mutations (Kabat EU numbering):

-L235W/G236N/H268D/Q295L/A330K/K326T (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pi) to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

SEQ ID NO: 102 SG1099 - Generic human heavy chain constant region derived from IgG1 containing mutations (Kabat EU numbering):

-Q311R/P343R (suitable for increasing pI to enhance antigen uptake)

SEQ ID NO: 103 SGI 100 generic human heavy chain constant region derived from IgG1 containing mutations (Kabat EU numbering):

-Q311R/P343R (suitable for increasing pI to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

CNTO888//11K2-DT IgG1 (type bispecific CNT0888//11K2-DT IgG1 Crossmab)

SEQ ID NO: 104 heavy chain 1-CNTO888//11K2-DT IgG1

SEQ ID NO: 105 heavy chain 2-CNTO888//11K2-DT IgG1

SEQ ID NO: 106 light chain 1-CNTO888//11K2-DT IgG1

SEQ ID NO: 107 light chain 2-CNTo888//11K2-DT IgG1

CKLO2 - IgG1 (type bispecific CKLO2 IgG1 Crossmab)

SEQ ID NO: 108 heavy chain 1- CKLO2 IgG1

SEQ ID NO: 109 heavy chain 2- CKLO2 IgG1

SEQ ID NO: 110 light chain 1- CKLO2 IgG1

SEQ ID NO: 111 light chain 2- CKLO2 IgG1

CKLO2 - SG1095 (generic bispecific CLOK2 Crossmab containing SG1095 Fc mutations)

SEQ ID NO: 112 heavy chain 1- CKLO2 - SG1095

SEQ ID NO: 113 heavy chain 2- CKLO2- SG1095

SEQ ID NO: 114 light chain 1- CKLO2- SG1095

SEQ ID NO: 115 light chain 2- CKLO2 - SG1095

CKLO2 - SG1099 (generic bispecific CKLO2 Crossmab containing SG1099 Fc mutations)

SEQ ID NO: 116 heavy chain 1- CKLO2 - SG1099

SEQ ID NO: 117 heavy chain 2- CKLO2 - SG1099

SEQ ID NO: 118 light chain 1- CKLO2 - SG1099

SEQ ID NO: 119 light chain 2- CKLO2 - SG1099

CKLO2 - SGI 100 (generic bispecific CKLO2 Crossmab containing SGI 100 Fc mutations)

SEQ ID NO: 120 heavy chain 1- CKLO2 - SGI 100

SEQ ID NO: 121 heavy chain 2- CKLO2 - SGI 100

SEQ ID NO: 122 light chain 1- CKLO2 - SGI 100

SEQ ID NO: 123 light chain 2- CKLO2 - SGI 100

CKLO3 - SG1095 (generic bispecific CLOK3 Crossmab containing SG1095 Fc mutations)

SEQ ID NO: 124 heavy chain 1- CKLO3 SG1095

SEQ ID NO: 125 heavy chain 2- CKLO3 SG1095

SEQ ID NO: 126 light chain 1- CKLO3 - SG1095

SEQ ID NO: 127 light chain 2- CKLO3 - SG1095

CKLO3-SG1099 (generic bispecific CKLO3 Crossmab containing SG1099 Fc mutations)

SEQ ID NO: 128 heavy chain 1- CKLO3 - SG1099

SEQ ID NO: 129 heavy chain 2- CKLO3 - SG1099

SEQ ID NO: 130 light chain 1- CKLO3 - SG1099

SEQ ID NO: 131 light chain 2- CKLO3- SG1099

CKLO3-SG1100 (generic bispecific CKLO3 Crossmab containing SG1100 Fc mutations)

SEQ ID NO: 132 heavy chain 1- CKLO3 - SG1100

SEQ ID NO: 133 heavy chain 2- CKLO3 - SG1100

SEQ ID NO: 134 light chain 1- CKLO3 - SG1100

SEQ ID NO: 135 light chain 2- CKLO3 - SG1100

Additional anti-CCL2 antigen-binding fragments:

SEQ ID NO: 136 heavy chain variable domain VH 2F2

SEQ ID NO: 137 VL 2F2 light chain variable domain

SEQ ID NO: 138 VH heavy chain variable domain mouse 11K2 (=11K2m)

SEQ ID NO: 139 VL light chain variable domain mouse 11K2 (=HK2m)

SEQ ID NO: 140 heavy chain variable domain VH 1H11

SEQ ID NO: 141 VL 1H11 light chain variable domain

Typical CCL2 and homologues (without signal peptide):

SEQ ID NO: 142 human type CCL2 (MCP1) - wild type (wt)

SEQ ID NO: 143 typical human CCL2 (MCP1) - variant P8A

SEQ ID NO: 144 typical human CCL2 (MCP1) - variant T10C

SEQ ID NO: 145 human type CCL8 (MCP2) - wild type (wt)

SEQ ID NO: 146 typical human CCL8 (MCP2) - variant P8A

SEQ ID NO: 147 human type CCL7 (MCP3) - wild type (wt)

SEQ ID NO: 148 human type CCL13 (MCP4) - wild type (wt)

SEQ ID NO: 149 Cynomolgus macaque type CCL2 wild type (wt)

SEQ ID NO: 150 wild type (wt) mouse type CCL2

Additional typical heavy chain constant regions:

SEQ ID NO: 151 GG01 - Generic human heavy chain constant region derived from IgG1 containing mutations (Kabat EU numbering):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

SEQ ID NO: 152 GG02 Generic human heavy chain constant region derived from IgG1 containing mutations (Kabat EU numbering):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-M428L/N434A/Y436T (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

SEQ ID NO: 153 GG03 - Generic human heavy chain constant region derived from IgG1 (containing IgG1 allotype sequences) containing mutations (Kabat EU numbering):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

SEQ ID NO: 154 GG04 - Generic human heavy chain constant region derived from IgG1 (containing IgG1 allotype sequences) containing mutations (Kabat EU numbering):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-M428L/N434A/Y436T (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

CKLO2 - GG01 (type bispecific CLOK2 Crossmab containing Fc mutations GG01)

SEQ ID NO: 155 heavy chain 1- CKLO2 - GG01

SEQ ID NO: 156 heavy chain 2- CKLO2 - GG01

SEQ ID NO: 157 light chain 1- CKLO2 - GG01

SEQ ID NO: 158 light chain 2- CKLO2 - GG01

CKLO2 - GG02 (type bispecific CLOK2 Crossmab containing Fc mutations GG02)

SEQ ID NO: 159 heavy chain 1- CKLO2 GG02

SEQ ID NO: 160 heavy chain 2- CKLO2 GG02

SEQ ID NO: 161 light chain 1- CKLO2 GG02

SEQ ID NO: 162 light chain 2- CKLO2 GG02

CKLO2 GG03 (type bispecific CLOK2 Crossmab containing Fc mutations GG03)

SEQ ID NO: 163 heavy chain 1- CKLO2 - GG03

SEQ ID NO: 164 heavy chain 2- CKLO2 - GG03

SEQ ID NO: 165 light chain 1- CKLO2 - GG03

SEQ ID NO: 166 light chain 2- CKLO2 - GG03

CKLO2 - GG04 (type bispecific CKLO2 Crossmab containing Fc mutations GG04)

SEQ ID NO: 167 heavy chain 1- CKLO2 - GG04

SEQ ID NO: 168 heavy chain 2- CKLO2 - GG04

SEQ ID NO: 169 light chain 1- CKLO2 - GG04

SEQ ID NO: 170 light chain 2- CKLO2 - GG04

CKLO2 - GG03/GG04 (generic bispecific CKLO2 Crossmab containing GG03 Fc mutations in the knob chain and containing GG04 Fc mutations in the trench chain)

SEQ ID NO: 171 heavy chain 1- CKLO2 - GG03/GG04

SEQ ID NO: 172 heavy chain 2- CKLO2 - GG03/GG04

SEQ ID NO: 173 light chain 1- CKLO2 - GG03/GG04

SEQ ID NO: 174 light chain 2- CKLO2 - GG03/GG04

Designation of the monospecific, unmodified anti-CCL2 antibodies/antigen-binding fragments used for the bispecific anti-CCL2 antibodies described herein

Designation of bispecific antibodies to CCL2 with unmodified VH/VL as Crossmab (see WO 2016/016299) with IgG1 or IgG1 containing mutations L234A, L235A and P329G (PGLALA)

Designation of VH/VL modified bispecific antibodies as Crossmab (see WO 2016/016299). Depending on the heavy chain constant domain used (e.g. wild type IgG1, PGLALA, SG1095, SG1099, 1100), the suffixes wild type IgG1, PGLALA, SG1095, SG1099, 1100 are added

Examples:

Example A-1 monospecific antibodies to CCL2

Generation of monospecific antibodies to CCL2 and CCL2 antigen

Recombinant DNA technologies

For DNA manipulation, standard methods were used as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Synthesis of genes and oligonucleotides

The required gene segments were obtained by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, the assembly of short synthetic DNA fragments was carried out by annealing chemically synthesized oligonucleotides or by PCR. The corresponding oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany)

Description of a basic/standard mammalian expression plasmid

To express the desired gene/protein (e.g., full-length antibody heavy chain, full-length antibody light chain, or CCL-2 molecule), a transcription unit containing the following functional elements was used:

- immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

- 5'-untranslated region (5'UTR) of the heavy chain of human immunoglobulin,

- signal sequence of mouse immunoglobulin heavy chain,

- gene/protein to be expressed (e.g. full-length antibody heavy chain or antibody light chain or CCL-2 molecule) and

- bovine growth hormone polyadenylation sequence (BGH pA).

In addition to the expression unit/cassette containing the gene to be expressed, the basic/standard mammalian expression plasmid contains

the origin of replication from the pUC18 vector, which allows replication of this plasmid in E. coli, and

a beta-lactamase gene that confers ampicillin resistance in E. coli.

Generation of expression plasmids for recombinant monoclonal antibodies and CCL-2 molecules

Expression plasmids for transient expression of monoclonal antibodies and CCL-2 antigens contained, in addition to the corresponding expression cassettes, an origin of replication from the pUC18 vector, which ensures the replication of this plasmid in E. coli, and a beta-lactamase gene, which confers resistance to ampicillin in E. coli.

The transcription unit of the corresponding NS or LC immunoglobulin or CCL-2 molecule contained the following functional elements:

- immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

- 5'-untranslated region (5'UTR) of the heavy chain of human immunoglobulin,

- the signal sequence of the mouse immunoglobulin heavy chain and

- bovine growth hormone polyadenylation sequence (BGH pA).

The corresponding antibodies 1A4, 1A5, 1G9, 2F6, CNTQ888, murine and humanized 11K2, ABN912, based on their VH and VL, were created as wild-type IgG1 and as IgG1 PGLALA/silencing Fc-effector kappa light chain

Transient Expression and Purification

Recombinant production was performed by transiently transfecting HEK293 cells (derived from the human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). To obtain monoclonal antibodies, cells were cotransfected with plasmids containing the corresponding immunoglobulin heavy and light chains. Transfection reagent 293-fectin (Invitrogen) was used for transfection. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants were collected three to seven (3-7) days after transfection. Supernatants were stored at reduced temperature (eg -80°C).

General information regarding recombinant expression of human immunoglobulins, for example in HEK293 cells, is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden) and Superdex 200 size exclusion chromatography (GE Healthcare, Sweden). Briefly, sterile filtered cell culture supernatants were immobilized on MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 25 mM sodium citrate. at pH 3.0. The eluted protein fractions were pooled, neutralized with 2 M Tris, pH 9.0, and further purified by size exclusion chromatography using a Superdex 200 26/60 GL column (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0 . Size exclusion chromatography fractions were analyzed by CE-SDS (Caliper Life Science, USA), and antibody-containing fractions were pooled and stored at -80°C.

Generation of recombinant CCL2

Wild-type CCL-2 can exist as a monomer but can actually also form dimers at physiological concentrations. This equilibrium between monomers and dimers may vary and should be carefully considered for all described in vitro experiments in which different concentrations may be used. To avoid uncertainty, we generated CCL2 variants with point mutations: The P8A variant of CCL2 carries a mutation in the dimerization interface that results in an inability to form a dimer, resulting in a certain pure CCL2 monomer. In contrast, the T10C variant of CCL2 results in a fixed CCL2 dimer (J Am Chem Soc. 2013 Mar 20;135(11):4325-32).

The corresponding soluble CCL2 protein (wild type, P8A or T10C variants) was purified from cell culture supernatants by cation exchange chromatography using SP-Sepharose HP (GE Healthcare, Sweden) and Superdex 200 size exclusion chromatography (GE Healthcare, Sweden). Briefly, sterile filtered cell culture supernatants were diluted with 10 mM KH2PO4, pH 5.0 to bring conductance to <4 mS/cm. The diluted supernatant was loaded onto SP-Sepharose resin equilibrated with 10 mM KH2PO4, pH 5.0, washed with equilibration buffer, and eluted using a gradient to 10 mM KH2PO4, 1 M NaCl, pH 5.0. The eluted protein fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 16/60 GL column (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by SDS-PAGE and analytical high-performance size exclusion chromatography. Fractions containing CCL2 were pooled and stored at -80°C.

Functional characteristics (binding)

The T200 instrument was equipped with a CM5 Biacore Series S sensor chip. The system buffer was HBS-ET (10 mM GEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). The system was set to 37°C. For each measurement, the sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyldextran, Fluka).

An antibody capture system was installed. 14,000 GARFcy (goat anti-rabbit Fcγ), 111-005-046, Jackson ImmunoResearch) was immobilized at 25°C at 25 μg/ml in 10 mM sodium acetate buffer, pH 5.0, by EDC/NHS coupling according to the manufacturer's description . The capture system was restored at 20 μl/min by 15 s injection of HBS buffer (100 mM HEPES, pH 7.4, 1.5 M NaCl, 0.05% (w/v) Tween 20), 1 min injection 10 mM glycine buffer, pH 2.0, followed by two injections within 1 min of 10 mM glycine buffer, pH 2.25. In another embodiment, mouse monoclonal antibodies were captured on the biosensor by immobilizing 12700 PE polyclonal rabbit anti-mouse (RAMIgG, GE Healthcare) antibodies onto the CM5 Biacore Series sensor as described above. The sensor was restored by introducing 10 mM glycine buffer, pH 1.7, for 3 min.

Supernatants of antibody clones were diluted 1:2 in system buffer and captured for 1 min at 5 μl/min. After antibody capture, the system was washed with 2.5-fold concentrated system buffer for 30 s at 80 μl/min, followed by stabilization at baseline for 2 min. Analyte kinetics were performed at 30 μL/min. Human CCL2 wt or the monomeric variant of CCL2, CCL2 P8A, was used as an analyte in solution. Analytes were administered at the highest concentration of 90 nM. The analyte contact time was 3 min and the dissociation time was 10 min. Biaevaluation V.3.0 software was used according to the GEHC manufacturer's instructions. A 1:1 binding model with local RMAX was used to subjectively estimate kinetic rates.

Antibody binding to human wild-type (wt) CCL2 and CCL2 variant P8A (monomer)

Cumulative pH-dependent CCL2 binding kinetics obtained from SPR I assay

The T200 instrument was equipped with a CM5 Biacore Series S sensor chip. The system buffer was HBS-ET (10 mM GEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) P20). In other embodiments, the pH of the system buffer was adjusted to pH 8.3, pH 7.9, pH 7.4, pH 7.1, pH 6.7, pH 6.3, pH 5.9, pH 5.5. The system was set to 25°C. For each measurement, the sample buffer was system buffer supplemented with 1 mg/ml CMD (carboxymethyldextran, Fluka).

An antibody capture system was installed. 13,000 MAb<h-Fc-pan>M-R10Z8E9-IgG (Roche) was immobilized at 25°C at 18 μg/ml in 10 mM sodium acetate buffer, pH 5.0, by EDC/NHS coupling as described by the manufacturer. The capture system was reconstituted by injecting at 20 μL/min HBS buffer (100 mM HEPES, pH 7.4, 1.5 M NaCl, 0.05% (w/v) Tween 20) followed by 1 min. 15 with 10 mM NaOH and two injections of 10 mM glycine buffer, pH 2.5, for 1 min. Antibody capture and injection was carried out for 30 s at 10 μl/min at a concentration of 80 nM with dilution in the appropriate system buffer. After antibody capture, the system was washed with 2.5-fold concentrated system buffer for 30 s at 50 μl/min, followed by stabilization at baseline for 2 min. Concentration dependent analyte series were administered in 1:3 dilution steps from 0 nM (buffer control) 0.4 nM, 1.1 nM, two injections at 3.3 nM, 30 nM. The analyte contact time was 3 min and the dissociation time was 10 min. Analyte kinetics were performed at 50 μL/min.

The capture of human antibodies in the form of ligands was carried out on the sensor surface:

• Normal human IgG as a positive control (H-N-IgG, Id.: 11717570, Roche),

• mAb to human CCL2 (humanized 11k2: CCL2-0002),

• mAb to human CCL2 (AB912, CCL2-0003) and

• mAb to human CCL2 (CNT0888, CCL2-0004);

• system buffer as a negative control.

Biaevaluation V.3.0 software was used according to the GEHC manufacturer's instructions. A 1:1 binding model with local R _MAX was used to determine kinetic rates.

Cross-reacting homologues of CCL

Since CCL2 (MCP-1) has high homology to CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), and these CCL chemokines are able to bind to CCR2, the binding of antibodies to CCL2 to these homologues was assessed. The results are shown in Fig. 1. With the exception of CNT0888, which is described to have selectivity for CCL2 (Mol Immunol. 2012 Jun; 51(2):227-33), the other antibodies tested bound to CCL7 or CCL8 (showed cross-reactivity with CCL7 or CCL8).

Biacore Assay Method: Binding of anti-CCL2 antibodies to CCL homologs such as CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3) and CCL13 (MCP-4) was assessed at 25°C using the instrument Biacore T200 (GE Healthcare). Mouse anti-human IgG (Fc) antibody (GE Healthcare) was immobilized onto each flow cell of the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES buffer, pH 7.4 (20 mM ACES, 150 mM NaCl, 1 mg/ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibody capture was performed on anti-Fc touch surfaces, then recombinant human CCL homolog proteins were passed through the flow cell at 5 nM and 20 nM. Wild-type CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3), and CCL13 (MCP-4) were commercially available from R&D Systems, while the CCL2 monomer (variant P8A) was an in-house generated antigen. Touch surfaces were restored each cycle with 3 M MgCl2. Binding sensorgrams were processed using Biacore T200 version 2.0 evaluation software (GE Healthcare).

Functional characteristics (biological)

CCR2 Alarm I - Calcium Flux Analysis

THP-1 cells (human acute monocytic leukemia cell line; ATCC TIB-202) were cultured in RPMI 1640, 10% FBS, 1 mM sodium pyruvate, 10 mM GEPES, 50 μM β-mercaptoethanol (supplied by Thermo Fisher Scientific). On the day of analysis, the cell density was adjusted to 8.33 × 10 ⁵ cells/ml in 25.8 ml of analytical medium (RPMI 1640 without PBS). FLIPR® Calcium Assay Kits (FLIPR Calcium 4, Cat. No. R8142, Molecular Devices) were used for detecting intracellular calcium changes in a homogeneous assay format.

The dye loading solution was prepared by mixing two vials of component A with 20 ml of component B (HBSS buffer plus 20 mM HEPES, pH 7.4) according to the manufacturer's instructions, Molecular Devices. 516 μl of 1 M GEPES (final analytical concentration: 10 mM) was added, followed by 516 μl of 250 mM probenecid (final analytical concentration: 2.5 mM). For the stock solution, 65.4 mg of probenecid (Sigma P8761) was dissolved in 465 μl of 1 N. NaOH and 465 μl of 1x HBSS (Thermo Fisher Scientific) was added. 25.8 ml of loading buffer was mixed with 25.8 ml of assay medium with cells, which is enough for example for four microtiter plates (volume required 52.6 ml; 10 ⁶ THP-1/ml). 120 μl of cell suspension in loading buffer was transferred to each well of a 96-well F-bottom cell culture plate. The plates were incubated at room temperature for 3-4 hours.

At the same time, a solution of antibody and ligand was prepared. Eight concentrations of each antibody were tested from 30 μg/ml to 0.025 μg/ml (without serial dilution, final concentration in wells). Each concentration was tested in two plates. All dilutions were prepared in analytical medium as a 10-fold concentrated solution. Anti-human CCL2/JE/MCP-1 antibody (R&D Systems, cat. no. MAB279) was used as a reference antibody. CCL2 ligand (R&D Systems, cat. no. 279-MS-10) was prepared by dissolving 50 μg of CCL2 lyophilisate in 500 μl of RPMI 1640 (100 μg/ml) and transferring 400 μl into 10 ml of analytical medium (4 μg/ml stock solution) . Ionomycin (Sigma, cat. no. I-0634) (1 mg ionomycin dissolved in 1340 μl DMSO (Sigma, cat. no. D-8779), 1 mM) was used as a stimulation control. 10 μl of 1 mM stock solution was diluted in 1990 μl of analytical medium (5 μM, final analytical concentration 500 nM). 100 μl was pipetted into the appropriate control wells of polypropylene MTP.

Antibody and CCL2 dilutions were preincubated in two 96-well V-shaped polypropylene plates. 50 μl of 4 μg/ml CCL2 stock solution (final concentration 400 ng/ml CCL2) and 50 μl of a 10-fold concentrated antibody dilution were pipetted into the well. The plates were incubated for 30-60 minutes at room temperature.

After incubation, the cell plate and compound plate were transferred directly to the reading position of a FlexStation® 3 (Molecular Devices) and calcium assays were performed as described in the system instructions (excitation 485 nm, emission 525 nm). The data was read at intervals of several seconds.

Results:

Efficacy of anti-CCL2 antibodies in inhibiting CCL2-induced internalization of the CCR2 receptor expressed on monocytes

To prevent ligand-induced internalization of CCR2 on myeloid cells, we developed an in vitro assay and characterized anti-CCL2 antibodies. Monocytes were isolated from the peripheral blood of healthy donors by magnetic separation using a commercial kit (Stemcell, cat. no. 15068). To block monocytes were preincubated with normal human IgG (Privigen, CSL Behring) at a final concentration of 500 μg/ml on ice for 50 min in FACS buffer (PBS+0.2% BSA). Cells were then centrifuged for 10 min (300xg, 4°C), washed again with FACS buffer, and stored on ice. Dilutions of anti-CCL2 antibodies (50 μl each) were prepared (in parallel experiments at 4°C and 37°C) in 96 U-bottom (BD) wells. Monocytes were separated, resuspended in medium (RPMI 1640; 10% FBS; 2 mM L-glutamine) and incubated at 4°C and 37°C, respectively, until further use. Recombinant CCL2 (50 μl; final concentration 100 ng/ml) was added to prepared antibody dilutions (at different concentrations) at both 4°C and 37°C. 100 μl of monocyte suspension (2 x 10 ⁵ cells/well) was added to the CCL2/anti-CCL2 antibody mixture for a total volume of 200 μl and the cells were incubated at 4°C and 37°C for 1 h 30 min before centrifugation at 300xg, 4° WITH. From this point on, all steps were carried out with pre-chilled buffers: cells were washed with 250 μl of FACS buffer and additionally cells were stained against the CCR2 receptor (using a commercial CCR2-APC conjugate or an appropriate isotype control-APC according to standard FACS protocols: Aliquots were stained at 5 μl/ 106 cells CD 192 (CCR2) APC (BioLegend, No. 357208, clone K036C2/mIgG2a κ), as well as the corresponding isotype control antibody: 20 μl/10 ⁶ cells mIgG2a k APC BD Biosciences, No. 400222, clone MOPC-173).

Receptor expression was then analyzed on FACS Canto II and CCR2 internalization was calculated as follows:

• No internalization: Cells were analyzed in the absence of ligand incubation (rec. CCL2).

• 100% internalization: Maximum reduced level of CCR2 expression on cells that were pre-incubated with rec. CCL2

Inhibition of CCL2-mediated chemotaxis on human THP-1 cells

Migration of CCR2 ⁺ THP1 cells towards the CCL2 gradient was studied as follows. Monocyte THP1 cells (ATCC© TIB-202™) were cultured in RPM1 1640 medium (PAN, cat. No. P04-17500), supplemented with FBS and L-glutamine. Cells were normally passaged two to three times before use in the migration assay and then maintained overnight in medium containing reduced FBS (1.5% instead of 10% FBS). Cells were counted and incubated with 10 μg/ml normal human IgG (Invitrogen, cat. #12000; to block FcgR) for 15 minutes at room temperature.

At the same time, anti-CCL2 antibodies (and/or controls) were added to the bottom chamber of a 96-well HTS Transwell plate system (Corning, cat. #3386; pore size 3 μm) containing serum-free medium with 25 ng/ml rhCCL-2 ( R&D Systems, cat. No. 279-MS). The insert plates were then inserted into the lower chamber plate and 75 μl (1.5 x 10 ⁵ cells) of the above cell suspension (including the IgG block) with or without 5 μg/ml antibody/isotype was added to each insert. The plates were covered and incubated overnight at 37°C in a CO2 incubator (5% CO2).

The insert plate was removed and Cell-titer-glo substrate (Promega, cat. no. G758) was added to each well of the lower chamber plate to determine the viability of migrated cells. After incubation for 1 hour on a shaker at 300 rpm (cover plate sealed), 200 µl from each well was transferred to a black 96-well Microfluor plate (VWR, cat. no. 735-0527) and luminescence was measured (luminescence reader, e.g. Bio-Tek, Tecan). The fold change was calculated as the ratio between the number of cells migrated (Cell Titer Glo, TEL) with control IgG antibody and anti-CCL2 antibodies. Table 2 below shows the results for 5 to 10 repetitions per condition:

Assessment of sweeping of the human CCL2 immune complex with monospecific (one paratope) antibodies to CCL2 in mice

To evaluate the ability of single-paratope antibodies to form an immune complex with wild-type human CCL2, preformed immune complexes consisting of single-paratope anti-CCL2 antibody (20 mg/kg) and wild-type human CCL2 (0.1 mg/kg) were administered in a single dose of 10 ml/kg into the tail vein of mice transgenic for human FcRn (B6.Cg-Fcgrt ^tm1Dcr Tg(FCGRT)32Dcr/DcrJ, Jackson Laboratory). Blood was collected at 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration. Serum was obtained by immediately centrifuging the blood at 14,000 rpm for 10 minutes at 4°C. Serum was stored at or below -80°C until measurement. The single paratope antibodies studied are listed in Table 3 below. Antibodies with Fc SGI have Fc gamma receptor binding similar to wild type, while antibodies with Fc SGI05 are characterized by silencing of Fc gamma receptor binding.

The effect of sweeping the immune complex of each anti-CCL2 antibody with one paratope on hCCL2 clearance in vivo was assessed by comparing the anti-CCL2 antibody with Fc-gamma receptor binding (SGI,=wild-type IgG1 with Fc-gamma receptor binding intact; solid line) and the antibody to CCL2 with Fc gamma receptor binding silencing (SG105, =IgG1 without Fc gamma receptor binding; dashed line), as shown in FIG. 2. The corresponding figures 2a to 2g present the serum concentration of hCCL2 over time after injection of preformed immune complexes consisting of hCCL2 and the corresponding anti-CCL2 antibody (with two different Fc moieties: SGI=wild type IgG1 with intact Fc-gamma binding -receptor and SG105=IgG1 without Fc-gamma receptor binding) in FcRn transgenic mice. Antibody profiles were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/Certara). PPKbesk was estimated using the linear logarithmic trapezoidal rule with extrapolation to infinity. Clearance values were determined as dose/AUC. This difference in clearance was also expressed as a fold change, which was calculated by dividing the clearance of hCCL2 Fc gamma receptor binding antibodies (SG1) by the clearance of hCCL2 Fc gamma receptor binding silencing antibodies (SG105) (Table 3 below). Data from Table 3 show that clearance of human CCL2 by Fc gamma receptor binding antibodies (SGI=wild type IgG1 with intact Fc gamma receptor binding) was similar to clearance by Fc gamma receptor binding silencing antibodies (SG105, without Fc-gamma receptor binding) for all antibodies studied with one paratope. This suggests that immune complex-mediated sweeping of CCL2 by the single-paratope antibodies tested was not effective.

Measuring total human serum CCL2 concentration using electrochemiluminescence (ECL)

The concentration of total human CCL2 in mouse serum was measured by ECL. 3 μg/ml anti-CCL2 antibody (F7 (Biolegend) or clone MAB679 (R&D Systems)) was immobilized on a 96-well MULTI-ARRAY plate (Meso Scale Discovery) overnight before incubation in blocking buffer for 2 hours at 30°C . Anti-CCL2 MAB679 was used as a capture antibody for samples containing humanized antibodies 11K2, 1A4 or 1A5. Anti-CCL2 clone 5D3-F7 was used for samples containing antibodies ABN912, CNTO888, 1G9, 2F6H. Human CCL2 calibration curve samples, quality control samples, and mouse serum samples were prepared by dilution in dilution buffer and incubation with excess drug for 30 minutes at 37°C. Samples were then added to the anti-CCL2 antibody-immobilized plate and allowed to bind for 1 hour at 30°C before washing. Next, SULFO TAG NHS ester-labeled (Meso Scale Discovery) anti-human Fc antibody (clone: JDC-10, SouthernBiotech) was added and the plate was incubated for 1 hour at 30°C before washing. Reading buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal was recorded using a SECTOR Imager 2400 (Meso Scale Discovery). Human CCL2 concentration was calculated from the response of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Measuring serum anti-CCL2 antibody concentration using enzyme-linked immunosorbent assay (ELISA)

The concentration of anti-CCL2 antibody in mouse serum was measured using ELISA. Anti-human IgG kappa chain antibody (Antibody Solutions) was spread onto a Nunc MaxiSorp plate (Thermofisher) and allowed to sit overnight at 4 degrees C to prepare immobilized anti-human IgG plates. The calibration curve and samples were prepared with 1% pooled mouse serum. The samples were then distributed onto anti-human IgG immobilized plates and allowed to settle for 1 hour at 30 degrees C. Goat anti-human IgG (gamma chain specific) with HRP conjugate (Southern Biotech) was then added to react for 1 hours at 30 degrees C. The chromogenic reaction was carried out using TMB substrate (Life Technologies) as the substrate. After stopping the reaction with 1 N. sulfuric acid (Wako) absorbance was measured at 450 nm on a microplate reader. Mouse plasma concentrations were calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Assessment of immune complex sweeping of endogenous murine CCL2 with a single paratope antibody in mice

In addition to the above results (which suggest that immune complex-mediated CCL2 sweeping of the single-paratope antibodies tested was not effective), further evaluation was performed.

To evaluate the ability of single-paratope antibodies to form and clear an immune complex with endogenous murine CCL2, mice were injected with mouse cross-reacting single-paratope anti-CCL2 antibodies 11K2. Humanized 11K2H2-SG1 (wild type IgG1 = Fc gamma receptor binding) or humanized 11K2-SG105 (Fc gamma receptor binding silencing) antibodies were administered intravenously at a single dose of 20 mg/kg at a single dose of 10 ml/kg in the tail vein Balb/c mice. Blood was collected before administration, 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration. Serum was obtained by immediately centrifuging the blood at 14,000 rpm for 10 minutes at 4°C. Serum was stored at or below -80°C until measurement.

In FIG. 3 shows the time course of serum concentrations of total murine CCL2 and the antibody-time profile for humanized 11K2-SG1 and 11K2-SG105 in mice.

As can be seen in FIG. 3, levels of accumulated murine CCL2 did not differ between 11K2-SG105 (Fc gamma receptor binding silencing Fc) and 11K2-SG1 (wild type IgG1 = Fc gamma receptor binding Fc). This shows that Fc gamma receptor-mediated clearance of endogenous murine CCL2 by administered antibodies was absent or negligible. Antigens in the immune complex are cleared faster than non-complexed antigens due to multimeric recruitment of Fc-gamma receptors, suggesting that the 11K2 antibody was not able to form immune complexes with endogenous murine CCL2.

Measuring the concentration of murine CCL2 in mouse serum using enzyme-linked immunosorbent assay (ELISA)

Mouse CCL2 concentrations in mouse serum were measured by adapting reagents from a commercially available murine CCL2 ELISA kit (R&D Systems). The manufacturer's protocol was followed except for the preparation of samples for the calibration curve. Purified recombinant mouse CCL2 was used as a standard instead of the manufacturer's protein. For samples obtained after antibody administration, calibration curve samples and samples were prepared with 2.5% mouse serum injected antibody diluted at 40 micrograms/ml and incubated for 30 minutes at 37 degrees C. Samples were then distributed on plates with immobilized antibody to human CCL2 and incubated at 30 degrees C for 2 hours. Detection was performed by adding mouse MCP-1 conjugate and incubating at 30 degrees C for 2 hours, followed by the addition of substrate and stop solution.

For samples obtained before antibody administration, the Mouse MCP-1 Ultrasensitive Kit (Meso Scale Discovery) was used according to the manufacturer's instructions. The antibody was not dissolved in the sample before being added to the plate.

Measuring serum anti-CCL2 antibody concentration using enzyme-linked immunosorbent assay (ELISA)

The concentration of anti-CCL2 antibody in mouse serum was measured using ELISA. Anti-human IgG kappa chain antibody (Antibody Solutions) was spread onto a Nunc MaxiSorp plate (Thermofisher) and allowed to sit overnight at 4 degrees C to prepare immobilized anti-human IgG plates. The calibration curve and samples were prepared with 1% pooled mouse serum. The samples were then distributed onto anti-human IgG immobilized plates and allowed to settle for 1 hour at room temperature. HRP-conjugated mouse anti-human IgG (clone JDC-10, Southern Biotech) was then added to react for 30 minutes at room temperature. The chromogenic reaction was carried out using ABT substrate (KPL) as the substrate, and the absorbance at 405 nm was measured using a microplate reader. Mouse plasma concentrations were calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Conclusion on various mouse PK studies with monospecific (one paratope) antibodies to CCL2

Based on the summary results of mouse PK studies, none of the single paratope antibodies tested demonstrated effective clearance of CCL2 from the circulation. These data suggest that single-paratope antibodies are unable to form an immune complex with CCL2 for its efficient clearance from the circulation.

In contrast, as described below, bispecific anti-CCL2 antibodies with two different antigen-binding moieties/sites (two-paratope anti-CCL2 antibodies) were able to efficiently form immune complexes with CCL2 and clear it from circulation.

Example B-1

Bispecific (two paratope) antibodies to CCL2

Several bispecific antibodies to CCL2 were generated with two different antigen binding fragments (paratopes) binding to two different specific epitopes on human CCL2

Introduction

To investigate whether single antigen binding or cross-linking has a significant impact on in vivo clearance of CCL2, we generated bispecific CCL2 antibodies with 2 different antigen-binding moieties/sites that bind to 2 different epitopes on CCL2 using bispecific CrossMab Technology (see e.g. WO 2009/080252, WO 2015/150447), WO 2009/080253, WO 2009/080251, WO 2016/016299, Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20) (bispecific (= with two paratopes) CrossMab). These molecules were first studied in vitro for their biochemical and functional properties, but they also served as tools to evaluate in vivo clearance of CCL2 in a murine co-administration study. To evaluate clearance potential based on Fc gamma receptor (FcgR) binding-mediated sweeping (eg, Igawa et al, Immunological Reviews 270 (2016) 132-151, WO 2012/122011, WO 2016/098357 and WO 2013/ 081143), we generated all Crossmabs as wild-type huIgG1, which binds to FcgR, and modified human IgG1 constant chain, which have reduced/eliminate binding to FcgR effector-silencing molecules (e.g., IgG1 with mutations L234A, L235A, P329G (EU numbering according to Kabat).

Identification of suitable anti-CCL2 antibody pairs - Selection of two-paratope antibodies using sandwich ELISA.

Sandwich ELISA was performed to identify pairs of antibodies that do not compete for binding to human CCL2. MAXISORP 384-well plates (NUNC) were coated with 1 μg/ml of the 7 indicated capture antibodies (group 1) and blocked with 2% BSA. Biotinylated (NHS-PEO ₄ -Biotin, Pierce) human CCL2 WT (20 ng/ml) was incubated with an excess amount of the same 7 antibodies (group 2) at 1 μg/ml or blocking buffer for 1 hour at 37 degrees Celsius. After incubation, the mixtures were added to a blocked ELISA plate and incubated for 1 hour at room temperature. Detection of plate-bound CCL2 was performed using streptavidin-HRP followed by the single-component substrate TMB (Lifetech). The signal was stopped with 1 n. acid HCl (Wako). O.P. wells without competition antibody were taken as 100% signal for each capture antibody. O.P. empty wells without the addition of CCL2 were taken as 0% signal. Nine pairs of antibodies that did not show strong competition for binding to CCL2 in either direction were selected as candidates for the generation of Crossmab bispecific antibodies.

Creation and study of the characteristics of antibodies to CCL2 with two paratopes and immune complexes

Generation of anti-CCL2 antibodies with two paratopes in the bispecific CrossMab format using recombinant DNA technology

For DNA manipulation, standard methods were used as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Synthesis of genes and oligonucleotides

The required gene segments were obtained by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, the assembly of short synthetic DNA fragments was carried out by annealing chemically synthesized oligonucleotides or by PCR. The corresponding oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany)

Description of a basic/standard mammalian expression plasmid

To express the desired gene/protein (e.g., antibody heavy chain or antibody light chain), a transcription unit containing the following functional elements was used:

• immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

• 5'-untranslated region (5'UTR) of the heavy chain of human immunoglobulin,

• signal sequence of mouse immunoglobulin heavy chain,

• gene/protein to be expressed (eg full length antibody heavy chain or light chain antibody or MHC class I molecule) and

• bovine growth hormone polyadenylation sequence (BGH pA).

• In addition to the expression unit/cassette containing the gene to be expressed, the basic/standard mammalian expression plasmid contains

• the origin of replication from the pUC18 vector, which allows replication of this plasmid in E. coli, and

• Beta-lactamase gene that confers ampicillin resistance in E. coli.

Creation of expression plasmids for recombinant monoclonal antibodies

Recombinant monoclonal antibody genes encode the corresponding immunoglobulin heavy and light chains.

Expression plasmids for transient expression of monoclonal antibody molecules contained, in addition to an immunoglobulin heavy or light chain expression cassette, an origin of replication from the pUC18 vector, which ensures the replication of this plasmid in E. coli, and a beta-lactamase gene, which confers resistance to ampicillin in E. coli.

The transcription unit of the corresponding antibody heavy or light chain contained the following functional elements:

• immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

• 5'-untranslated region (5'UTR) of the heavy chain of human immunoglobulin,

• signal sequence of mouse immunoglobulin heavy chain,

• the corresponding cDNA sequence of the heavy or light chain of the antibody and

• bovine growth hormone polyadenylation sequence (BGH pA).

Temporal Expression and Analytical Characteristics

Recombinant production was performed by transiently transfecting HEK293 cells (derived from the human embryonic kidney cell line 293) cultured in F17 medium (Invitrogen Corp.). To obtain monoclonal antibodies, cells were cotransfected with plasmids containing the corresponding immunoglobulin heavy and light chains. Transfection reagent 293-fectin (Invitrogen) was used for transfection. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants were collected three to seven (3-7) days after transfection. Supernatants were stored at reduced temperature (eg -80°C).

General information regarding recombinant expression of human immunoglobulins, for example in HEK293 cells, is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203. To create the following bispecific antibodies, the CrossMab technology described in WO 2016/016299 was used, in which a VH/VL exchange was carried out in one antibody arm, and the CH1/CL contact surface of the other antibody arm was modified through charge modifications, in combination with the "knobs" technology into the depressions" in the CH3/CH3 contact surface to stimulate heterodimerization. A typical sequence for all four antibody chains when using this technology is given in CNTO888//11K2-DT IgG1 (see SEQ ID NO: 104 to SEQ ID NO: 107)

List of Crossmab anti-CCL2 bispecific (two-paragon) antibodies generated with wild-type IgG1 (IgG1 WT) (Wild-type IgG1 means without modifications/mutations that affect Fc receptor binding, and includes heterodimerization technology such as "knobs to trenches" ")

List of created bispecific (with two paratopes) Crossmab antibodies to CCL2 with IgG1 containing Fc-gamma receptor silencing mutations L234A, L235A, P329G (EU numbering according to Kabat) (IgG1 -PGLALA)

Purification of anti-CCL2 antibodies with two paratopes

Dual-paratope anti-CCL2 antibody-containing cell culture supernatants were filtered and purified through up to three chromatographic steps. Depending on the purity of the eluate from the capture step, an ion exchange chromatography step was optionally applied between the capture step and final purification.

Dual paratope anti-CCL2 antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden), POROS 50 HS (Thermofisher Scientific) and Superdex 200 size exclusion chromatography (GE Healthcare, Sweden). Briefly, sterile filtered cell culture supernatants were immobilized on MabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 25 mM sodium citrate. at pH 3.0. The eluted protein fractions were pooled and neutralized with 2 M Tris, pH 9.0. Ion exchange chromatography as an optional purification step was performed with POROS 50 HS (Thermofisher Scientific), equilibration and wash with 20 mM histidine, pH 5.6, and loading of the diluted eluate from the capture step was performed with 20 mM histidine, 0.5 M NaCl at pH 5 ,6. Fractions from ion exchange chromatography were analyzed by CE-SDS LabChip GX II (PerkinElmer) and Crossmab-containing fractions were pooled. Size exclusion chromatography on Superdex 200 (GE Healthcare) was used as a second or third purification step. Size exclusion chromatography was carried out in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by CE-SDS LabChip GX II (PerkinElmer), and Crossmab-containing fractions were pooled and stored at -80°C.

In case of satisfactory product quality after POROS 50 HS (ThermoFisher Scientific), size exclusion chromatography on Superdex 200 (GE Healthcare) was replaced by desalting chromatography on HiPrep 26/10 Desalting (GE Healthcare) in 20 mM histidine buffer, 0.14 M NaCl, pH 6 ,0. Protein concentration for antibody preparations was determined by measuring optical density (OD) at 280 nm using a molar extinction coefficient calculated from the amino acid sequence. Antibody purity and integrity were analyzed by CE-SDS LabChip GX II (PerkinElmer) with Protein Express Chip and HT Protein Express Reagent Kit. The content of antibody drug aggregates was determined by high-throughput SEC using a Biosuite high-resolution SEC, 250 Å, 5 μm analytical size exclusion column (Waters GmbH), using 200 mM K2HPO4/KH2PO4, 250 mM KCl, pH 7.0, as a running buffer. Average purity was 94-100% by CE-SDS analysis and monomer content was >95% (EC).

Functional characteristics of bispecific (with two paratopes) antibodies to CCL2

Affinity (binding) measurement

Approximately 1200 resonance units (RU) of a capture system (20 μg/mL goat anti-human Fc IgG; order code: 109-005-098; Jackson Immuno Research) were coupled to a C1 chip (GE Healthcare BR-1005-35) at pH 5.0 using the amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween 20), pH 7.4. The flow cell temperature was set to 25°C and the sample block temperature was set to 12°C and primed twice with running buffer. Bispecific antibody capture was performed by injecting 2 μg/mL solution over 60 s at a flow rate of 10 μL/min. The association was measured by injecting various concentrations of human CCL2 (wt) into solution for 150 s at a flow rate of 30 μL/min, starting at 30 nM with 1:10 dilutions. The dissociation phase was monitored for up to 1200 s and initiated by switching from sample solution to running buffer. The surface was restored by rinsing for 60 seconds with 0.85% H ₃ PO ₄ solution at a flow rate of 10 μl/min. The volume refractive index was corrected by subtracting the response obtained from the goat anti-human IgG Fc-coated surface. Control administrations were also subtracted (= double matching). To calculate the kinetic parameters, the 1:1 Langmuir model was used.

Natural formation of an immune complex in the presence of a wild-type antigen.

All protein samples (CrossMab anti-CCL2 bispecific antibodies and antigens) were re-buffered in 1x PBS, pH 7.4, using dialysis or centrifugal ultrafiltration devices.

Serial dilutions of CrossMab samples were prepared from 2.0 to 0.1 mg/mL. Similarly, antigen solutions were prepared in PBS with concentrations ranging from 0.012 to 0.23 mg/ml. Concentrations were chosen so that equivalent volumes could be mixed to provide a constant molar ratio of 1:1 (antibody:CCL2 complex). The antigen used in this study was wild type CCL2.

Equivalent volumes of pre-diluted CrossMab and CCL2 preparations were mixed and incubated at 37°C for 1 hour before applying samples to a Superose6 column (GE Healthcare, #2039) pre-equilibrated with PBS and eluting at a flow rate of 0.5 ml/min. A total of 100 μg or the maximum possible volume of 250 μl was used, and antibodies and antigens separately were used as controls.

SEC-MALLS data were recorded using an OptiLab rEX refractive index detector and a miniDAWN Treos MALLS detector (both from Wyatt inc.). SEC-MALLS signals were processed using Astra V5 software (Wyatt).

CCR2 reporter assay to investigate the neutralization characteristics of anti-CCL2 antibodies

Tango™ CCR2-bla U20S cells were purchased from Invitrogen, Germany, to study the effect of CCL2 neutralizing antibody constructs. These reporter cells contain human chemokine (C-C motif) receptor 2 (CCR2) coupled to the TEV protease site and the transcription factor Gal4-VP16, stably integrated into the parental Tango™ GPCR-bla U2OS cell line. This parental cell line stably expresses a beta-arrestin/TEV protease fusion protein and a beta-lactamase (bld) reporter gene under the control of a UAS response element. Addition of natural ligands MCP1=CCL2 resulted in measures of reporter gene activity that can be measured by cleavage of the FRET-activated substrate.

In principle, the analysis and cell processing procedures were performed according to the supplier's manual. Briefly, CCR2-U20S cells were seeded at a density of 2 × 10 ⁴ cells/well (96-well black clear-bottom plate, cat. no. 655090, Greiner Bio-one) in 50 μl of assay medium (Freestyle 293 expression medium, cat. no. 12338-018, Invitrogen). In parallel, serial dilutions of various antibodies under study were prepared, as well as a solution of the CCL2 antigen at c = 4x final concentration. CCL2 antigen/antibody mixtures were then prepared and pre-incubated for two to three hours (h) at RT. 50 μl of the indicated CCL2/antibody solutions were transferred into CCR2-expressing U2OS cells and incubated for 18 hours in a humidified incubator at 37°C and 5% CO ₂ . One analytical medium was used as a control.

The next day, CCF4 substrate (cat. no. K1089, Invitrogen) with β-lactamase loading solution (cat. no. K1085, Invitrogen) was prepared and added to the cells at 20 μl/well. The substrate solution was incubated for two hours at RT in the dark.

Finally, fluorescence wavelengths were determined using a Spectra Max (M4) reader (Molecular devices) at the following wavelengths (Exc./Use = 409 nm/460 nm = blue*; Ex./Use = 409 nm/530 nm = green**), and the blue/green fluorescence ratio after subtracting the assay control was calculated according to the following equation: ratio = (sample-blue* - control-blue*)/ (sample-green** - control-green** ).

Following pH engineering, we examined the characteristics of the final LO candidates (CKLO1–4) with respect to their ability to inhibit CCL2-induced CCR2 signaling. In this case, the neutralization property was assessed only by the monomeric version of the rivers. CCL2 protein, which was used at a final concentration of ca. 15 ng/ml.

Assessment of sweeping of the human CCL2 immune complex with a dual paratope antibody in mice

To evaluate the ability of dual-paratope antibodies to form an immune complex with wild-type human CCL2, preformed immune complexes consisting of dual-paratope anti-CCL2 (20 mg/kg) and wild-type human CCL2 (0.1 mg/kg) were administered in a single dose of 10 ml/kg into the tail vein of Balb/c mice. Blood was collected at 5 minutes, 7 hours, 1 day, 3 days and 7 days after administration. Serum was obtained by immediately centrifuging the blood at 14,000 rpm for 10 minutes at 4°C. Serum was stored at or below -80°C until measurement. The two paratope antibodies studied are listed in Table 4 below. Antibodies with Fc IgG1 WT have Fc-gamma receptor binding similar to wild type, whereas antibodies with Fc PGLALA are characterized by silencing of Fc-gamma receptor binding. The results are shown in Fig. 4a-4k.

The effect of immune complex sweeping of each anti-CCL2 antibody with two paratopes on hCCL2 clearance in vivo was assessed by comparing the anti-CCL2 antibody with Fc-gamma receptor binding (solid line) and the anti-CCL2 antibody with Fc-gamma receptor binding silencing (PGLALA; dotted line) ), as shown in Fig. 4a-4k. Antibody profiles were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/Certara). PPKbesk was estimated using the linear logarithmic trapezoidal rule with extrapolation to infinity. Clearance values were determined as dose/AUC. This difference in clearance was also expressed as fold change, which was calculated by dividing the clearance of hCCL2 Fc gamma receptor binding antibodies (SG1) by the clearance of hCCL2 Fc gamma receptor binding silencing antibodies (PGLALA) (Table 4 below). Data from Table 4 below indicate that clearance of human CCL2 by Fc gamma receptor (FcgR) binding antibodies (IgG1 WT) was superior to binding by Fc gamma receptor binding silencing antibodies (PGLALA, with IgG1 Fc domain containing L234A mutations , L235A, P329G (EU numbering according to Kabat)) for all antibodies with two paratopes studied. This suggests that the immune complex-mediated sweep of CCL2 mediated by the dual paratope antibodies tested was more efficient. In addition, several antibodies with two paratopes showed large fold changes in clearance values, for example, CNTO//humanized 11K2 (CNTO//11K2).

The fold change is calculated by dividing the clearance of hCCL2 antibodies with FcgammaR (FcgR) binding WT by the clearance of hCCL2 antibodies with PGLALA. As shown in FIG. 4A-4k and Table 4 below, CNTO//11k2 exhibits the highest fold change of 21.5 among the wild type (WT) IgG1 antibody that has FcgR binding and the FcgR binding silencing antibody (PGLALA). This suggests that the immune complex-mediated CNTO//11k2-WT IgG1 sweep is the most effective among all variants.

Measuring total human serum CCL2 concentration using electrochemiluminescence (ECL)

The concentration of total human CCL2 in mouse serum was measured by ECL. 3 μg/ml anti-CCL2 antibody 2F2-SG1 was immobilized on a 96-well MULTI-ARRAY plate (Meso Scale Discovery) overnight before incubation in blocking buffer for 2 hours at 30°C. Human CCL2 calibration curve samples, quality control samples, and diluted mouse serum samples were incubated with denaturing buffer consisting of 9% SDS for 30 minutes at 37°C or glycine-HCl buffer pH 2.0-2.5 for 10 minutes at 37°C. The purpose of using a denaturing buffer was to dissociate human CCL2 from the dual paratope antibody. Samples were then diluted 10-fold, added to a plate containing immobilized anti-CCL2 antibody, and allowed to bind for 1 hour at 30°C before washing. SULFO TAG-labeled MCP-1 antibody was then added and the plate was incubated for 1 hour at 30°C before washing. Reading buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal was recorded using a SECTOR Imager 2400 (Meso Scale Discovery). Human CCL2 concentration was calculated from the response of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Measuring serum anti-CCL2 antibody concentration using enzyme-linked immunosorbent assay (ELISA)

The concentration of anti-CCL2 antibody in mouse serum was measured using ELISA. Anti-human IgG kappa chain antibody (Antibody Solutions) was spread onto a Nunc MaxiSorp plate (Thermofisher) and allowed to sit overnight at 4 degrees C to prepare immobilized anti-human IgG plates. The calibration curve and samples were prepared with 1% pooled mouse serum. The samples were then distributed onto anti-human IgG immobilized plates and allowed to settle for 1 hour at 30 degrees C. Goat anti-human IgG (gamma chain specific) with HRP conjugate (Southern Biotech) was then added to react for 1 hours at 30 degrees C. The chromogenic reaction was carried out using ABTS substrate (KPL) as the substrate, and the absorbance was measured at 450 nm using a microplate reader. Mouse plasma concentrations were calculated from the absorbance of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Research results

Based on the summary results of these murine PK studies, sweeping of human CCL2 with the tested two-paratope antibodies was more effective compared to single-paratope antibodies at the same dose. For single paratope antibodies, a minimal difference in antigen clearance was observed between the FcgR binding WT and FcgR binding silencing antibodies (Table 3). In contrast, a large difference in antigen clearance was observed between dual paratope antibodies with WT FcgR binding and FcgR binding silencing (Table 4), suggesting that the dual paratope antibodies tested can efficiently sweep CCL2. The combination CNT0888//11K2 was chosen for additional antibody engineering because it demonstrated the largest fold change in clearance.

Example B-2 Antibodies to CCL2 with modified variable domains and CDR (ion-dependent/pH-dependent binding)

Modification leading to ion-dependent/pH-dependent binding

To generate pH-dependent antibodies to CCL2, histidine scanning mutagenesis was performed on all CDRs of mAb CNT0888 and humanized 11K2. Each amino acid in the CDR was individually replaced with histidine using the In-Fusion HD cloning kit (Clontech Inc. or Takara Bio company) according to the manufacturer's instructions. After confirming by sequencing that each variant was correctly mutated, variants were transiently expressed and purified using the following method: Recombinant antibodies were transiently expressed using Freestyle FS293-F and 293Fectin cells (Life technologies), according to the manufacturer's instructions. Recombinant antibodies were purified with protein A (GE Healthcare) and eluted in D-PBS or His buffer (20 mM histidine, 150 mM NaCl, pH 6.0). If necessary, additional size exclusion chromatography was performed to remove high and/or low molecular weight components. All histidine substitution variants were assessed using a modification of the BIACORE® assay described above. Briefly, an additional dissociation phase at pH 5.8 was integrated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. This was done to evaluate the pH-dependent dissociation between antibody (Ab) and antigen (Ag) from complexes formed at pH 7.4 compared to the corresponding dissociation at pH 5.8. The dissociation rate in pH 5.8 buffer was determined by processing and fitting the data using Scrubber 2.0 curve fitting software (BioLogic Software).

Single histidine substitutions that resulted in a decrease in the binding response at pH 5.8 compared to the dissociation phase at pH 7.4 were selected and pooled. To identify mutations that improved affinity at pH 7.4, more than 500 variants were generated for the heavy and light chain, respectively, using at least one variant generated by the histidine substitution step. In these variants, each amino acid in the CDR was replaced by 18 other amino acids, excluding the original amino acid and cysteine. The ability of variants to bind to human CCL2 was assessed at 37 degrees C at pH 7.4 using a BIACORE® 4000 instrument (GE Healthcare). As before, an additional dissociation phase at pH 5.8 was integrated into the BIACORE® assay immediately after the dissociation phase at pH 7.4. The dissociation rate in pH 5.8 buffer was determined by processing and fitting the data using Scrubber 2.0 curve fitting software (BioLogic Software).

Variants that had improved affinity at pH 7.4 and improved pH sensitivity were selected and these mutations were combined. This is illustrated by the four 11K2 variants and four CNT0888 variants in the table below.

To evaluate the combined effect of modified 11K2 variants and CNT0888, each 11K2 variant was combined with four modified CNT0888 variants and expressed as a dual-paratope anti-CCL2 antibody in CrossMab format. This is illustrated in the table below, where the 4x4 combination results in the creation of 16 antibodies with two paratopes, designated CKLO01 - CKLO16.

To create bispecific antibodies, the CrossMab technology described in WO 2016/016299 was used, in which a VH/VL exchange was carried out in one arm of the antibody, and the CH1/CL contact surface of the other arm of the antibody was modified through charge modifications, in combination with the overhang technology depressions" in the CH3/CH3 contact surface to stimulate heterodimerization. A typical sequence for all four antibody chains when using this technology is given in CKLO2 IgG1 (see SEQ ID NO: 108 to SEQ ID NO: 111). Depending on the heavy chain constant domain used (e.g. wild type IgG1 (without Fc receptor binding silencing mutations), PGLALA, SG1095, SG1099, 1100 in the case of SG1095, SG1099, 1100 see description below or sequence description) suffixes IgG1, PGLALA are added , SG1095, SG1099, 1100.

Functional characteristics of anti-CCL2 antibodies with two paratopes with modified variable domains and CDR (non-dependent/pH-dependent binding)

Affinity measurements (see methods above)

For all 16 wild-type IgG1 bispecific antibodies generated against CCL2, their pH-dependent binding to human CCL2 was determined.

In FIG. Figure 5a shows Biacore® sensorgrams showing the binding profile to monomeric CCL2 at pH 7.4 (black line) and pH 5.8 (gray line) of four modified 11K2 and four CNT0888 variants, as well as 16 Crossmabs after combination.

In FIG. 56 shows Biacore® sensorgrams showing the binding profile of four modified 11K2 and four CNT0888 variants, as well as 16 Crossmabs after combination, with monomeric CCL2 when an additional dissociation phase at pH 5.8 was integrated into the BIACORE® assay immediately after the dissociation phase at pH 7.4.

Cross-linking with CCL8

pH-dependent binding to recombinant monomeric human CCL2 and recombinant monomeric human CCL8 was assessed at 37°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc antibody (GE Healthcare) was immobilized onto each flow cell of the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES buffer, pH 7.4 or pH 5.8 (20 mM ACES, 150 mM NaCl, 1 mg/ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibody capture was performed on anti-Fc touch surfaces, then recombinant monomeric human CCL2 was passed through the flow cell at a concentration of 8 nM. The association phase of the analytes with antibodies was monitored for 120 s, followed by the dissociation phase for 180 s. Touch surfaces were restored each cycle with 3 M MgCl ₂ . The binding sensorgram was processed using TIBCO Spofire by normalizing the binding response relative to the capture level.

The assessment of pH-dependent dissociation for antibody/antigen complexes formed at pH 7.4 was tested using a modified Biacore assay. Briefly, an additional dissociation phase at pH 5.8 was integrated into the Biacore assay immediately after the dissociation phase at pH 7.4. The binding sensorgram was processed using TIBCO Spofire by normalizing the binding response relative to the capture level.

Expression and purification of recombinant human CCL8 P8A monomer: The wild-type human CCL8 sequence was obtained from Genbank (NCBI: NP_005614.2). To create monomeric CCL8, the proline at position 8 of the mature CCL8 protein was replaced with alanine. Expi 293 cells (Lifetech) were transfected according to the manufacturer's instructions. Wild-type CCL8 and monomeric P8A protein were purified using the same method from cell culture supernatants by cation exchange chromatography using SP-Sepharose HP (GE Healthcare) and Superose 200 size exclusion chromatography (GE Healthcare). Briefly, cell culture supernatants were diluted 2.5-fold with MilliQ water (Millipore), loaded onto a Hi-Trap SP-HP column equilibrated with PBS, washed with equilibration buffer, and eluted using a 0 2 M NaCl gradient. The eluted protein fractions were pooled and further purified by size exclusion chromatography using a HiLoad 16/600 Superose 200 column (GE Healthcare) equilibrated with 20 mM histidine, 150 mM NaCl, pH 6.0. Fractions were analyzed by size exclusion chromatography and SDS-PAGE. Fractions containing CCL8 protein were pooled, concentrated and stored at -80°C.

Human CCL8 has high homology to CCL2 and is also capable of binding to CCR2. Because the 11K2 arm is capable of binding to CCL8 (see Fig. 1), it was necessary to identify mutations to reduce this binding to avoid possible off-target effects of CCL8 neutralization. In addition, elimination of CCL8 binding by the 11K2 arm is important for efficient immune complex formation with CCL2. Because the CNTO arm does not bind to CCL8, binding of CCL8 to the 11K2 arm will interfere with immune complex formation with CCL2, which may reduce the clearance of CCL2 from plasma.

To identify mutations that reduce 11K2 binding to human CCL8 and confer selectivity for human CCL2, some CDR positions were replaced, such as D101E in 11K2 VH CKLO02 or W92R in 11K2 VL CKLO03, to eliminate cross-reactivity with huCCL8. As shown in FIG. 6, the binding of CCL8 Crossmab to two paratopes can be significantly reduced by engineering 11K2. The CKLO01 variant was not optimized to reduce CCL8 binding, whereas CKLO04, CKLO03, and CKLO02 contain mutations to reduce CCL8 binding. All four Crossmabs exhibit pH-dependent binding to CCL8.

Binding affinity of anti-CCL2 antibodies to recombinant CCL2 and CCL8 at pH 7.4 and pH 5.8

To determine the affinity and pH-dependent binding of parental CNT0888H/11K2H2, CKLO1, CKLO2 and CKLO3 to human CCL2 and CCL8, assays were performed at 37°C using a Biacore T200 instrument (GE Healthcare). Anti-human Fc antibody (GE Healthcare) was immobilized onto each flow cell of the CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the manufacturer's recommended settings. Antibodies and analytes were diluted in ACES buffer, pH 7.4 or pH 5.8 (20 mM ACES, 150 mM NaCl, 1 mg/ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibody capture was performed on anti-Fc touch surfaces, then the recombinant human CCL2 P8A variant (monomer) or the CCL8 P8A variant (monomer) was passed through the flow cell at concentrations ranging from 1.25 nM to 20 nM, obtained by two-fold serial dilution. Touch surfaces were restored each cycle with 3 M MgCl2. Binding affinity was determined by processing and fitting the data 1:1 to a binding model using Biacore T200 Evaluation software version 2.0 (GE Healthcare). The binding affinities of anti-CCL2 antibodies to recombinant CCL2 and CCL8 at pH 7.4 and pH 5.8 are shown in Table 5 below.

The data in Table 5 show that binding to CCL8 at pH 7.4 was reduced in the case of CKL02 and CKL03, while maintaining strong affinity at pH 7.4 and pH-dependent binding in the case of CCL2. The results show that various modifications made to the variable regions and CDRs of the parent bispecific antibody based on CNT0888 and 11K2 resulted in the successful generation of affinity matured variants CKLO1, CKLO2, CKLO3 with increased binding affinity to CCL2 compared to the parent Ab at pH 7. 4. At the same time, CKLO1, CKLO2, CKLO3 demonstrated strong pH-dependent binding to CCL2. A more than 1000-fold lower KD of binding affinity for CCL2 at pH 5.8 was observed compared to the KD value at pH 7.4.

Clearance of wild-type human CCL2

To evaluate the ability of pH-dependent bispecific antibodies to increase the clearance of wild-type human CCL2, preformed immune complexes consisting of a single paratope anti-CCL2 antibody (20 mg/kg) and wild-type human CCL2 (0.1 mg/kg) were injected into a single dose of 10 ml/kg into the tail vein of SCID mice. Blood was collected at 5 minutes, 1 hour, 4 hours, 7 hours, 1 day and 7 days after administration. Serum was stored at or below -80°C until measurement. The Crossmab antibodies studied were the parent CNTO//11K2 and four pH engineered variants CKLO01, CKLO02, CKLO03 and CKLO04. All antibodies contained the Fc portion of wild-type IgG1 (without Fc (gamma) receptor binding/silencing mutations). Measurements of total human CCL2 and anti-CCL2 antibody concentrations in mouse serum were performed as described above (in the section entitled “Evaluation of Sweeping of Human CCL2 Immune Complex with Dual Paratope Antibody in Mice” and Table 4).

The results are shown in Fig. 7a: Serum concentration of hCCL2 over time after injection of a preformed immune complex consisting of hCCL2 and bispecific antibodies to CCL2 (parental CNTO//11K2 and pH-dependent variants CKLO01, CKLO02, CKLO03 and CKLO04) into SCID mice. All four pH-engineered variants demonstrated rapid clearance of human CCL2. In the case of CKLO02, CKLO03, CCL2 was below the detection limit on day 1. In the case of the parent CNTO//11K2, rapid clearance of human CCL2 was initially observed until day 1, but thereafter clearance of human CCL2 was slow.

Example B-3 Antibodies to CCL2 with modified variable domains and CDR (ion-dependent/pH-dependent binding) and Fc-mediated sweeping

Modification of bispecific antibodies to CCL2 using sweep technology

The CCL2 bispecific antibodies have been modified using sweep technology so that the CCL2 bispecific antibodies can reduce the amount of free CCL2 over large periods of time to allow long-lasting biological effects such as anti-cancer efficacy or anti-inflammatory efficacy in vivo.

The concept of sweeping is described, for example, in Igawa et al, Immunological Reviews 270 (2016) 132-151, WO 2012/122011, WO 2016/098357 and WO 2013/081143, which are incorporated herein by reference.

pI Fc-mediated sweeping

Having demonstrated that pH-engineered antibodies with two paratopes can accelerate CCL2 clearance in vivo, we next assessed the ability of antibodies with pI-increasing substitutions to enhance the clearance of wild-type human CCL2. Preformed immune complexes consisting of single-paratope anti-CCL2 antibody (20 mg/kg) and wild-type human CCL2 (0.1 mg/kg) were injected in a single dose of 10 ml/kg into the tail vein of SCID mice. Blood was drawn at 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 7 days after administration. Serum was stored at or below -80°C until measurement. The Crossmab antibodies studied were parent CKLO03 with IgG1 and CKLO03 with Fc with increased pI, CKLO03-SG1099. Measurement of total human CCL2 and anti-CCL2 antibody concentrations in mouse serum was performed as described above (in the section entitled “Evaluation of Sweeping of Human CCL2 Immune Complex with Dual-Paratope Antibody in Mice” and Table 4).

The results are shown in Fig. 7b: Serum concentration of hCCL2 over time after injection of a preformed immune complex consisting of hCCL2 and CKLO03 (with wild-type IgG1 Fc) or CKLO03-SG1099 (CKLO03 with elevated pI Fc) into SCID mice. CKLO03-SG1099, which contains substitutions in Fc Q311R/P343R (Kabat EU numbering), showed faster clearance/reduction of human CCL2 compared to CKLO03 with IgG1. This demonstrates that a pH-dependent dual paratope antibody with pI-increasing mutations can accelerate CCL2 clearance.

Generation of anti-CCL2 antibodies with two paratopes with FcgammaRIIb-enhanced Fc variants and other Fc modifications

This example illustrates the engineering of Fc to enhance CCL2 clearance.

In WO 2013/125667 it was demonstrated that the clearance of soluble antigen can be increased by introducing antigen binding molecules (eg antibodies) containing an Fc domain exhibiting increased affinity for FcgammaRIIb. In addition, Fc variants that exhibit increased binding to human FcgammaRIIb have been illustrated in WO 2012/115241 and WO 2014/030728. It has also been demonstrated that these Fc variants may exhibit selectively increased binding to human FcgammaRIIb and decreased binding to other Fc gamma receptors (FcgammaR). This selective increase in FcgammaRIIb binding may be beneficial not only for soluble antigen clearance, but also for reducing the risk of unwanted effector functions and immune response.

To develop an antibody drug, it is necessary to evaluate the efficacy, pharmacokinetics and safety in non-human animals in which the drug is pharmacologically active. If it is active only in humans, alternative approaches such as the use of a surrogate antibody should be considered (Int. J. Tox. 28: 230-253 (2009)). However, it will not be easy to accurately predict the effects of interaction between the Fc region and FcgammaR in humans using a surrogate antibody, since the expression profiles and/or function of FcgammaR in non-human animals are not always the same as in humans. It would be preferable for the Fc regions of antibody therapeutics to be cross-reactive with non-human animals, particularly cynomolgus monkeys, which share similar FcgammaR expression and function profiles with humans, so that results obtained in non-human animals can be could be extrapolated to humans.

The following variants of the constant domain/Fc IgG1 of bispecific antibodies to CCL2 with mutations in the positions of the Fc part were created (EU numbering according to Kabat)

SG1095 - derived from IgG1, containing mutations (EU numbering according to Kabat):

-L235W/G236N/H268D/Q295L/A330K/K326T (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

SG1099 - derived from IgG1, containing mutations (EU numbering according to Kabat):

-Q311R/P343R (suitable for increasing pI to enhance antigen uptake)

SG1100 - derived from IgG1, containing mutations (EU numbering according to Kabat):

-Q311R/P343R (suitable for increasing pI to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

GG01 - derived from IgG1, containing mutations (EU numbering according to Kabat):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

GG02 - derived from IgG1, containing mutations (EU numbering according to Kabat):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-M428L/N434A/Y436T (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

GG03 derived from IgG1 (allotype SG1-IgG1), containing mutations (EU numbering according to Kabat):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-N434A (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

GG04 - derived from IgGl (allotype SG1-IgG1), containing mutations (EU numbering according to Kabat):

-L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing affinity for human FcgRIIb and decreasing affinity for other human FcgRs);

-Q311R/P343R (suitable for increasing isoelectric point (pI) to enhance antigen uptake);

-M428L/N434A/Y436T (suitable for increasing affinity for FcRn to prolong the plasma half-life of the antibody); And

-Q438R/S440E (suitable for suppression of rheumatoid factor binding)

Functional characteristics of bispecific antibodies to CCL2 with modified variable domains and CDR (ion-dependent/pH-dependent binding) and with or without Fc-mediated sweeping

SPR binding of Fc variants CKLQ2 SG1095. GG01. GG02.GG03/04

In a Biacore 8K SPR assay, the binding of monomeric human and cynomolgus CCL2 to 4 different antibodies P1AD8325, P1AF8137, P1AF8139 and P1AF8140 was examined at pH 7.4 and 5.8.

In this case, human Fab-kappa CaptureSelect™ (ThermoFisher Scientific) was immobilized on a CM3 sensor chip using the amine conjugation method, different antibodies were captured as ligands, and measurements were performed at concentrations of 0, 10, 100, and 1000 monomeric human and cynomolgus CCL2 nM as an analyte at two different pH values.

CKLO2-SG1095, CKLO2-GG01, CKLO2-GG02, CKLO2-GG03/04 show almost identical binding profiles to human and cynomolgus CCL2, which binds at 10, 100 and 1000 nM and dissociates equally quickly at pH 7.4, whereas at pH 5.8 no stable binding was observed. The results are shown in the table below.

Chemotactic analysis

Description of the method

THP-1 cells were cultured for 3 days to 8x10E5 cells/ml. A total cell number of 5000 cells/well was seeded in the upper chamber of a microtiter plate and allowed to settle at 37°C. Recombinant huCCL2 was pipetted into the lower chamber at a final concentration of 50 ng/ml in the presence or absence of anti-CCL2 antibodies (when the assay was performed to examine surrogate antibodies, muCCL2 at 100 ng/ml was used instead). The upper and lower chambers were brought together to avoid the formation of bubbles, and the plate was then incubated at 37°C for 24 hours. Migrated cells were quantified using the Cell Titer Glo method according to the manufacturer's recommendations and luminescence was measured using a Tecan Infinite 200 reader.

The results are shown in Fig. 8: Chemotactic assay: Bispecific antibodies to CCL2 with identical CDR and VH/VL variable regions, namely wild-type CKLO2-IgG1 and CKLO2-SG1095, but with different Fc fragments, can inhibit THP-1 cell migration with identical potency (IC ₅₀ =0.2 μg/ml; Fig. 8, left panel).

Similarly, CCL2-0048, the parent VH/VL-unmodified bispecific antibody CNT0888/11k2 from CKLO2, which is not pH-sensitive, also exhibits an IC ₅₀ of 0.2 μg/ml, since pH dependence is critical for antigen sweeping, a phenomenon which is not observed in this analysis.

The corresponding single paratope antibodies CNTO888 IgG1 and humanized 11k2 IgG1 showed IC ₅₀ values of 0.3 and 0.7 μg/ml, respectively, while the isotype control huIgG1 showed no inhibition (Figure 8, right panel).

In additional similar experiments, the IC ₅₀ values for CKLO2-GG01 (0.2 μg/ml), CKLO2-GG02 (0.2 μg/ml) and GG03/GG04 (0.3 μg/ml) were determined.

In vivo biological activity in a genetically modified mouse model

Material and methods

Model B16-huCCL2/CCL2-zero

This model was created to study antibodies to human CCL2 without the influence of murine CCL2 in otherwise competent tumor-bearing mice. The mouse tumor cell line B16-F10 was chosen for this purpose because it does not secrete mCCL2 and has been shown to grow in vivo in C57/B16 mice, the genetic background for CCL2 knockout mice.

To generate stable pools of B16F10 tumor cells expressing huCCL2, they were transfected with plasmid DNA encoding huCCL2 and the hygromycin-B selection cassette. For this purpose, cells were seeded in 6-well plates with 2.0E+05 cells/well in growth medium (DMEM + 10% FBS + 2 mM L-glutamine). After 24 h, a transfection mixture consisting of 1 μg of DNA per well and Lipofectamine 2000 in Opti-MEM medium was added to the cells. After this, the cells were subjected to selection conditions with a medium containing hygromycin-B (0.5 mg/ml). After 20 days of culture, live single cells were sorted based on FSC/SSC scattering using a BD FACS Aria III. Twenty days later, cell culture supernatants from single-cell clones were screened for human CCL2 expression using the Ready-SET-Go ELISA from ebioscience (cat. no. 88-7399-86) against wild-type B16F10 cells (data not shown).

Selected tumor cell clones B16-F10_HOMSA_CCL2 1A5, 2AZ and 2B2 were routinely cultured in DMEM containing 10% FBS and 2 mM L-glutamine (PAN Biotech GmbH, Germany) at 37°C in a water-saturated atmosphere with 5% CO2. The culture was passaged using trypsin/EDTA 1x (PAN Biotech GmbH, Germany), dividing twice a week.

Female B6.129S4-Ccl2tmlRol/J mice (Jackson Laboratories), 7-10 weeks old at the time of admission, were inoculated with B16-F10_HOMSA_CCL2 tumor cell clones: on this day (day 0 of the study), tumor cells were collected from culture flasks and transferred to culture medium, one washed once and resuspended in PBS. Cell numbers were determined using a cell counter and analyzer system (Vi-CELL, Beckman Coulter). For subcutaneous injection, the cell titer was adjusted to 1 × 10E7 cells/ml and 100 μl was injected subcutaneously into the right side of mice using a cooled

1.0 ml tuberculin syringe (Dispomed, Germany) and a small needle (0.45×12 mm). Cell inoculation was performed under general anesthesia with isoflurane (CP Pharma, Germany) in a small animal inhalation apparatus.

Tumor growth was monitored daily and mice were sacrificed on day 15 of the study, when tumors reached approximately 1000 mm3 for the B16-F10_HOMSA_CCL2 1A5 and 2A3 tumor cell clones (2B2 tumors were approximately 600 mm3 at this time point due to their slower growth rate). At the final time point, blood samples were collected for CCL2 measurements, and tumors were extracted and analyzed by flow cytometry as described above.

Murine immune cells were found to infiltrate all tumors, suggesting that human CCL2 is able to attract murine CCL2+ cells. The B16-F10_HOMSA_CCL2 1A5 tumor cell clone showed the largest CD45+ total infiltrate with the largest relative composition of mMSCs (monocytic myeloid suppressor cells) (Figure 2). Clones 2A3 and 2B2 had lower frequencies of immune cells in the tumors, although 2A3 cells resulted in similar levels of total serum CCL2 as 1A5 cells, whereas clone 2B2 exhibited significantly lower serum CCL2 concentrations (data not shown).

Female B6.129S4-Ccl2tm1Rol/J mice were inoculated with the B16-F10_HOMSA_CCL2 1A5 tumor cell clone as described above.

Treatment of study groups began 5 days after cell inoculation. Group 1 received human IgG vehicle control treatment, while groups 2 and 3 were treated daily with IP Mab CKLO2-IgG1 (wild-type IgG1 Fc) and CKLO2-SG1099 ((CKLO2 Fc with increased pI based on IgG1 with mutations Q311R7P343R (EU numbering according to Kabat), respectively, at 3.7 mg/kg for 9 days. On the day of the study, 14 mice were sacrificed and tumors were removed. Enzymatic digestion and cell dyes were used to create single-cell suspensions from each tumor mass for separate analysis by flow cytometry. The following markers and fluorochromes were used to identify immune cell populations of interest: CD45-BUV395, CD11b-BUV737, F4/80-BV421, CD11c-BV605, Ly6C-AF488, Ly6G-PerCP-Cy5.5, CD206-BV711, CD4- BV510, CD8a-APC-H7, NK1.1-PE-Cy7, CD279-APC, and CD274-PE Sample data were acquired on a BD LSR-Fortessa flow cytometer and analyzed using BD Diva software.

Serum samples were collected on days 6, 8, 11, and 14 to measure total and free huCCL2.

This method of detecting CCL2 is described in detail in the section entitled “Proof-of-Concept Study for the Efficiency of CCL2 Sweeping in Cynomolgus Macaques” below. For the analysis of free human CCL2 in this study, recombinant cynomolgus CCL2 was replaced with recombinant human CCL2 to prepare calibrators and QCs.

Serum samples of total CCL2 were analyzed using an unvalidated but eligible specific sandwich ELISA. Briefly, biotinylated anti-CCL2 capture antibody (CNT0888 CCL2-0004), blocking buffer, pretreated test sample, and detection reagent (digoxygenylated anti-CCL2 antibody (M-1H11-IgG)) were stepwise added to a 384-well streptavidin-coated microtiter plate and incubated on a low-power shaker for 1 hour at each step. To dissociate CCL2-drug complexes in samples from the pretreatment step, calibrators and QCs were acidified at pH 5.5 at 37°C for 10 minutes. Acidified samples were added to SA-MTP. To detect immobilized immune complexes, polyclonal antibody conjugate to digoxigenin POD was added and the plate was incubated for 60 minutes. After each step, the plate was washed three times to remove unbound substances.

ABTS was added to the plate and incubated at room temperature with shaking. Absorbance was measured at a wavelength of 405/490 nm. Human CCL2 concentrations were calculated from the response of the calibration curve using XLFit analytical software (IDBS).

Depending on the statistically analyzed data sets, the t test or one-way ANOVA with Tukey's test for multiple comparisons was used, respectively.

results

At the end of the study, tumor volume and tumor weight were significantly reduced in mice treated with CKLO2-SG1099 (CKLO2 with Fc with increased pI) (Figure 9). Closer analysis of the tumor infiltrate revealed a decrease in the tumor infiltrate of monocytic myeloid suppressor cells (M-MSCs), as expected after CCL2 blockade (Figure 9).

In addition, serum analysis confirmed the effectiveness of the therapy: pI optimization did result in reduced accumulation of total CCL2 compared to the CKLO2 molecule with wild-type IgG1 Fc, while free CCL2 (not bound by antibody) was completely suppressed below the detection limit (Fig. 10). Therefore, this model is suitable for studying the effects of blocking CCL2 in the context of a tumor using a sweep antibody to huCCL2 with two paratopes CKLO2-SG1099 (CKLO2 with Fc with increased pI). Subsequent studies will investigate the optimal dosing schedule in which CKLO2-SG1099 (CKLO2 Fc with increased pI) is administered at a lower dose once or twice weekly and the prolongation of free CCL2 suppression is monitored over weeks. Additionally, combination with T-cell activating therapy (ie, bispecific T-cell antibodies, PD-L1 blockade) is also being explored in this model.

In additional similar experiments, other variants such as CKLO2-SG1095, CKLO2-GG01, GG02 and GG03/GG04 are analyzed.

A proof-of-concept (POC) study of the effectiveness of CCL2 sweeping in cynomolgus monkeys

Methods. The primary objective of this study was to evaluate the extension of CCL2 suppression and sweeping efficiency of four anti-CCL2 antibodies (MCP-1) in cynomolgus monkeys. The secondary objective was to evaluate the pharmacokinetic (PK) properties of these antibodies. All antibodies were administered as single IV infusions of 25 mg/kg over a 30-minute period to 3 4-year-old male animals, and serum total CCL2 and antibody concentrations were measured for 70 days. The CCL2 antibodies tested consisted of control antibodies (groups 1 and 2) as well as antibodies specifically designed to provide enhanced drug clearance by the CCL2 complex (hereinafter referred to as antigen sweeping or simply sweeping). Group 1: CNTO888-SG1 (= wild-type IgG1) anti-CCL2 antibody (n=3 animals) as a control for maximal accumulation of total CCL2; group 2: anti-CCL2 antibody with two paratopes CKL02-SG1 (wild-type IgG1) with pH-dependent target binding, but without Fc modifications (n=3); group 3: anti-CCL2 antibody with two paratopes CKLO2-SG1100 with pH-dependent target binding and additional Fc-pI modifications (n=4) and group 4: anti-CCL2 antibody with two paratopes CKLO2-SG1095 with pH-dependent target binding, with Fc-pI and FcγRII and additional modifications (n=4).

This study quantified total serum concentrations of antibodies (free and antibody-bound CCL2) and free target. In addition, the presence of anti-drug antibodies (ADA) was assessed. Antibody, total and free CCL2 profiles were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/Certara); data were illustrated using GraphPad Prism v. 6.07 (GraphPad Software).

For antibodies, serum samples were analyzed using the general sandwich ELISA method for human proteins. Total antibody concentrations in monkey serum were measured by ELISA. 2 μg/ml anti-human kappa chain antibody was immobilized on a maxisorp 96-well plate overnight before incubation in blocking buffer for 2 hours at 30°C. Calibration curve antibody samples, quality control samples, and monkey serum samples were incubated in the plate for 1 hour at 30°C before washing. HRP-conjugated anti-human IgG antibody was then added and incubated for 1 hour at 30°C before washing. The ABTS substrate was incubated for 10, 20, and 30 min before detection using a 405 nm microplate reader. Antibody concentrations were calculated from the response of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Serum samples of total CCL2 were analyzed using an unvalidated but criteria-compliant ECL-specific sandwich assay. 3 μg/ml anti-CCL2 antibody (r2F2-SG1) was immobilized on a 96-well MULTI-ARRAY plate (Meso Scale Discovery) overnight before incubation in blocking buffer for 2 hours at 30°C. Simian CCL2 calibration curve samples, quality control samples, and diluted cynomolgus serum samples were incubated with acidic buffer, pH 5.5, for 10 minutes at 37°C. Samples were then incubated in a plate with immobilized anti-CCL2 antibody for 1 hour at 30°C before washing. SULFO TAG-labeled MCP-1 antibody was then added and incubated for 1 hour at 30°C before washing. Reading buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and the signal was recorded using a SECTOR Imager 2400 (Meso Scale Discovery). Cynomolgus CCL2 concentrations were calculated from the response of the calibration curve using SOFTmax PRO analysis software (Molecular Devices).

Serum samples of free CCL2 were analyzed using the unvalidated but eligible GyrolabTM immunoassay on the Gyrolab Xplore. Biotinylated anti-CCL2 antibody (M-2F6-IgG) was used as the capture reagent, and A1exa647-labeled anti-CCL2 antibody (M-1H11-IgG) was selected for detection. Both reagents were diluted to 1 μg/ml in PBS, 0.1% Tween, 1% BSA and transferred to a 96-well PCR plate (Fisher Scientific). Simian CCL2 samples for the calibration curve, QC, and undiluted serum samples were also transferred to a 96-well PCR plate. Both plates were loaded into the instrument along with a 200 nL Gyrolab Bioaffy disk (Gyros Protein Technologies AB). A three-step assay protocol (200-3 W-001) was selected. Briefly, the protocol describes the sequential addition of capture reagent, sample, and detection reagent to the designated streptavidin disk columns of the Gyrolab Bio Affy 200. Each reagent reaches the column at the same time after a short disk centrifugation step. After each step, the columns were washed with PBS 0.05% Tween and finally the laser-induced fluorescence values were recorded within the instrument. The concentration of free cynomolgus CCL2 was calculated from the response of the calibration curve using XL Fit software (IDBS).

ADA was analyzed using the method described elsewhere ( Stubenrauch et al., 2010 ). In general, biotinylated anti-human Fcγ-pan mAb R10Z8E9 was coupled to a streptavidin-coated high-binding plate at a concentration of 0.5 μg/ml and incubated for 1 h. Samples and standards were diluted with assay buffer to 5% cynomolgus serum and added to Each well of the coated plate was washed and incubated for 1 h with shaking. After washing, digoxygenylated anti-cynomolgus IgG antibody was added at 0.1 μg/ml and incubated for 1 h with shaking. After washing, a polyclonal anti-digoxigenin antibody-HRP conjugate was added at 25 mU/ml and incubated for 1 h with shaking. ABTS was added to the plate and incubated for 10 minutes at room temperature with shaking. Absorbance was measured on a microplate reader at a wavelength of 405/490 nm. ADA concentration was calculated from the response of the calibration curve using SOFTmax PRO analytical software (Molecular Devices).

Results. FC behavior was assessed during the time when animals were free of ADA (i.e., until day 14). During this period, serum concentration time profiles of all anti-CCL2 antibodies were similar (see Fig. 11, left panel) and partial mean AUC values (AUC _0-7d ) were comparable between the different groups, being 1490, 1810, 1210 and 1320 days/day. µg/ml for groups 1, 2, 3 and 4, respectively. Similarly, mean _Cmax values were comparable and were 620, 764, 616, and 664 μg/mL for groups 1, 2, 4, and 4, respectively. The extent of ADA occurrence varied widely between animals and groups, resulting in highly variable PK profiles after day 7 (data not shown). One animal from group 2 was ADA negative for the entire 70 day observation period (see FIG. 11, right panel). In this animal, the clearance, volume of distribution and terminal half-life of anti-CCL2 antibodies were estimated by non-compartmental analysis to be 7.34 ml(day.kg), 76.2 ml/kg and 10.9 days, respectively.

Baseline serum CCL2 levels were assessed for each animal before initiation of antibody treatment. Baseline CCL2 levels ranged from 0.126 to 0.357 ng/mL (geometric mean (%CV): 0.199 ng/mL (32.2%, N=14)). Because the free form of CCL2 has a faster clearance rate than the antibody-bound form of CCL2, an increase in total serum concentrations of CCL2 was expected following antibody treatment. This was indeed observed in all groups (Figure 12, left panel), demonstrating target binding by all antibodies. With treatment, the _Cmax values of total CCL2 increased to 824, 575, 106, 32.7 ng/ml for groups 1, 2, 3 and 4, respectively. AUC values _0-7d were 3060, 2970, 522 and 181 d.ng/ml for groups 1, 2, 3 and 4, respectively. While the animals were ADA negative, the molar concentration of drug remained in excess compared to the total concentration of CCL2. The two anti-CCL2 sweep antibodies (groups 3 and 4) demonstrated a significant reduction in serum concentrations of total CCL2 compared with the conventional antibody (group 1) by approximately 8- and 25-fold, respectively, based on _Cmax values and approximately 6-17-fold by based on AUC values of _0-7d for general CCL2. The ADA-negative animal from group 2 showed prolonged target binding (as seen by a plateau in total CCL2 concentration) (Fig. 12, right panel).

Treatment with all antibodies resulted in a significant reduction in serum total CCL2 levels (Figure 13, left panel), but the reduction was partially observed only initially. In group 1, all individuals again had quantifiable levels of free CCL2 after one day. In group 2, all individuals again had quantifiable levels of free CCL2 after two days. In groups 3 and 4, two individuals from each group showed suppression of free CCL2 for seven days. In group 3, two animals with moderate ADA responses, while maintaining sufficient antibody concentrations, showed suppression of free CCL2 below the detection limit for 21 days. In ADA-positive animals, antibody clearance was significantly increased and, as a consequence of decreased target binding, CCL2 levels quickly returned to baseline (not shown here).

Other variants such as CKLO2-SG1099, CKLO2-GG01, GG02, and GG03/GG04 are analyzed in additional similar experiments.

PK/PD study of the effectiveness of CCL2 sweeping in cynomolgus monkeys

Description and objectives of the study. The PK/PD study was designed based on the results of the PK study using the anti-CCL2 antibody CKL02-SG1095. Because the PK study demonstrated a high rate of anti-drug antibody (ADA) formation, treatment with Gazyvay® (obinutuzumab) was included in the PK/PD study to reduce the ADA response. To this end, doses of Gazyva 30 mg/kg were administered by intravenous infusion four times throughout the study: on days -14, -7, 8 and 36. CKL02-SG1095 was administered at dose levels of 2.5, 10 and 25 mg/kg to four animals (2/2 males and females) per dose group as an IV infusion over 30 minutes on day 1 (groups 1-3). For comparison, a conventional anti-CCL2 antibody (CNT0888-IgGl) was administered at a dose of 25 mg/kg as an IV infusion over 30 minutes on day 1 (group 4; same control as group 1 of the PC study described above). Total PK (CKL02-SG1095), total and free CCL2 concentrations were assessed until day 99 (ie, 14 weeks post-dose).

The objective of the PK/PD study was to demonstrate prolonged suppression of free CCL2 by CKL02-SG1095 compared with a conventional anti-CCL2 antibody (CNT0888 with wild-type IgG1 Fc portion) in non-human primates.

Methods. This study quantified total PK as well as total and free CCL2. However, due to the presence of Gazyvare in serum samples, some modifications were made in the total PK assay, total CCL2 assay, and ADA assay compared to the PC assay described herein. A PK assay has not been developed for CNTO888 IgG1.

The concentration of total antibody CKL02-SG1095 in monkey serum was measured by ELISA. For ELISA, biotinylated recombinant human CCL2 (antigen), pretreated test samples, positive control (calibrator) or QC (quality control) standards, and digoxygenylated anti-human IgG antibody (M-1.19.31-IgG) were sequentially added to a 384-well streptavidin-coated microtiter plate (SA-MTP). To dissociate the CCL2-drug complex, the test samples were pretreated at pH 5.5 for 20 minutes. Acidified samples were neutralized before addition to SA-MTP. Detection of immobilized immune complexes was carried out using a polyclonal antibody to digoxigenin-POD conjugate. After each step, the plate was washed three times to remove unbound substances. ABTS was added to the plate as a substrate and incubated at room temperature. Absorbance was measured at a wavelength of 405/490 nm. Antibody concentrations were calculated from the response of the calibration curve using XLFit analytical software (IDBS).

Serum samples of total CCL2 were analyzed using an unvalidated but eligible specific sandwich ELISA. Briefly, biotinylated anti-CCL2* capture antibody, pretreated test sample, and detection reagent (digoxygenylated anti-CCL2 antibody (1H11-IgG1)) were added stepwise to a 384-well streptavidin-coated microtiter plate and incubated on a low-power shaker for 1 hour in case capture and sample step and within 50 minutes in case of detection reagent, respectively. To dissociate CCL2-drug complexes in samples from the pretreatment step, calibrators and QCs were acidified at pH 5.5 for 20 minutes. Acidified samples were added to SA-MTP. To detect immobilized immune complexes, a polyclonal antibody to digoxigenin-POD conjugate was added and the plate was incubated for 50 minutes. After each step, the plate was washed three times to remove unbound substances.

ABTS was added to the plate and incubated at room temperature with shaking. Absorbance was measured at a wavelength of 405/490 nm. Cynomolgus CCL2 concentrations were calculated from the calibration curve response using XLFit analytical software (IDBS). *Capture antibody for Group 1 assay, Group 3: Anti-CCL2 CNTO8888 IgG1, Capture antibody for Group 4 assay: Anti-CCL2 2F2 IgG1.

ADA screening was performed using a bridge sandwich ELISA in 384-well plates. Animal samples from groups 1, 2 and 3, quality control samples and positive controls were incubated overnight with biotinylated capture antibody CKL02-SG1095 and digoxygenylated detection antibody CKL02-SG1095 along with two additional anti-CCL2 antibodies (2F6-IgG1 and 1H11 -IgG1) at RT, 500 rpm, on an MTP shaker; these antibodies were added to neutralize CCL2. For group 4 animal samples, biotin-labeled digoxygenylated CNT0888-SG1 was used, respectively. The formed immune complexes were transferred to streptavidin (SA)-coated MTP to immobilize the immune complexes using biotin (Bi)-labeled capture antibody. After aspiration of the supernatant, unbound substances were removed by repeated washing. Detection was performed by adding POD(p)-conjugated anti-digoxigenin antibody and ABTS substrate solution. The color intensity of the reaction was determined photometrically (absorbance at 405 nm - 490 nm reference wavelength). A sample was defined as ADA positive if the signal was found to exceed a specific cutoff point. This cutoff point was determined during assay qualification.

Results. Despite treatment with Gazyvay® (obinutuzumab), 10 of 12 animals treated with CKL02-SG1095 and 1 of 4 animals treated with CNT0888 developed ADAs that affected drug and biomarker concentrations. However, the two ADA-negative animals in the case of CKL02-SG1095 were in the 25 mg/kg dose group, allowing direct comparison with the ADA-negative animals in the CNT0888 group. FC behavior was assessed during the time when animals did not have ADA (i.e., until day 10). The PK profiles of the three different dose groups for CKL02-SG1095 are shown in Fig. 14, left panel. Partial mean AUC values (AUC _0-7d ) were 229/191 (male/female), 696/813, and 1492/1346 d.μg/mL for the 2.5, 10, and 25 mg/kg dose levels, respectively. C _max values were 115/122 (male/female), 369/491, and 869/941 μg/mL for the 2.5, 10, and 25 mg/kg dose levels, respectively. At the highest dose level, these results are consistent with the PC study. For two ADA-negative animals, the clearance, volume of distribution and terminal half-life of CKL02-SG1095 were estimated by non-compartmental analysis to be 10.5-17.4 ml/(day.kg), 116-118 ml/kg and 5.8- 11.6 days, respectively (see Fig. 14, right panel).

As for the PC study described above, accumulation of total CCL2 was observed after treatment with anti-CCL2 antibodies (see FIG. 15 left panel). Baseline CCL2 levels were assessed in five cases before drug administration (including a case before treatment with Gazyva® (obinutuzumab)); the mean baseline CCL2 value was 0.742 ng/mL, and treatment with Gazyva® (obinutuzumab) did not affect baseline CCL2 levels. The extent of total CCL2 accumulation (values in parentheses indicate median fold change relative to individual baseline levels) was dose- and design-dependent. In CKL02-SG1095, total CCL2 levels were increased to 22.4 (22), 67.2 (105) and 54.9 (76) ng/ml (median of four animals) for dose levels 2.5, 10 and 25 mg/kg. In the case of CNTO888 IgG1, the level of total CCL2 was increased to 1490 (3160) ng/ml (median of 4 animals). Comparison of ADA-negative animals from groups 3 and 4 showed significantly less accumulation for CKL02-SG1095 compared to CNT0888 (Fig. 15, right panel).

Treatment of all study groups resulted in a significant transient decrease in serum free CCL2 levels (see FIG. 16, left panel; generally below the detection limit (0.01 ng/ml). In all ADA-negative animals, free CCL2 levels quickly returned to baseline values after the onset of ADA (consistent with a decrease in drug exposure and rapid target relapse) In the case of ADA-negative animals (2/4 in group 3) and (3/4 in group 4), the duration of CCL2 suppression could be estimated at study time course. In the case of the conventional antibody CNT0888 (group 4), the duration of CCL2 suppression was short due to the intense accumulation of the common target (Fig. 15) Although drug concentration was not quantified (due to the lack of a specific assay available for CNT0888), PC study suggests similar PK properties between CNT0888 and CKL02-SG1095. On the other hand, prolonged suppression of free CCL2 was observed in the two ADA-negative animals from group 3. In one animal, CCL2 levels remained below the detection limit for 29 days (Figure 16, right panel).

Other variants such as CKLO2-SG1099, CKLO2-GG01, GG02, and GG03/GG04 are analyzed in additional similar experiments.

Study of the prevalence of CCL2 in different types of tumors

The following IHC study of the abundance of CCL2 and its receptor CCR2, including macrophage analysis using CD163/CD68 and CD14, was performed on 121 matched human tumor and serum samples for 6 different indications:

pancreatic cancer (PCa)

colorectal cancer (CRC)

breast cancer (BC)

prostate cancer (Prostate cancer)

ovarian cancer (OC)

stomach cancer (GC)

We were looking for answers to the following questions:

• Do these tumors (over)express CCL2?

• are CCL2 levels elevated in the blood of patients with these tumors?

• is there a correlation between blood and tumor levels of CCL2?

• is there a correlation between tumor CCL2 and infiltrating CCR2 ⁺ immune cells?

• is there a correlation between tumor CCL2 and infiltrating myeloid cells?

Material and methods

Histopathological evaluation was performed in a semi-quantitative manner. In addition, automated multiplex image analysis was used for CD163/CD68 IHC and assays were performed for CD14 and CCR2 for immune cell quantification, as well as for CCL2 quantification.

An immunohistological study was performed on a set of resection samples of 121 human tumors for 6 different indications: 31 pancreatic cancer (PCa), 30 colorectal cancer (CRC), 30 breast cancer (BC), 29 prostate cancer (PCa), 20 ovarian cancer ( GC) and 10 gastric cancer (GC) provided by Indivumed (Hamburg) and Asterand (Royston/Herts, UK). Tumors were fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned at 2.5 μm thickness, and mounted on Superfrost Plus slides. Mouse monoclonal antibody against CCL2 (clone 2D8, Novusbio NBP2-22115) was used on Ventana VXT following standard staining protocol (CC1 for 32', concentration 1 μg/ml in VBX, Optiview DAB detection system). A rabbit monoclonal anti-CCR2 antibody (E68, Abcam ab32144) was used on a Ventana Discovery XT following a standard staining protocol (CC1 for 32', 0.8 μg/mL concentration in DS2, Omni-UltraMap HRP DAB detection system). A mouse monoclonal antibody against the monocyte marker CD 14 (Cell Marque EPR3653, RTU) was used on the Ventana Discovery Ultra following a standard staining protocol (CC1 for 64', Omni-UltraMap HRP DAB Detection System). Double staining for macrophages and M2-like TAMs (tumor-associated macrophages) CD163/CD68 (DAB CD163 Mouse MRQ-26 Cell Marque RTU // red CD68 Mouse PG-M1 Dako) was used on Ventana VXT, following the standard staining protocol (CC1 for 32', CD163 RTU//CD68 concentration 0.6 μg/ml in DS2, detection using DAB and Red detection systems). All images were scanned using a Ventana iScan HT®. Tissue sections were analyzed in a semiquantitative manner.

1. Results

Prevalence of CCL2 and CCR2

All tumor indications analyzed demonstrated some tumors with CCL2 upregulation at different levels and the presence of CCR2 on TAMs in varying amounts (Table 6 below). For both CCL2 and CCR2, the highest expression was observed in ovarian carcinoma, followed by PDA and GC.

Tumor type-specific characteristics

Tumors with high CCL2-CCR2 activity associated with increased immunological tumor growth status with high MSC recruitment and M2 polarization are the preferred tumors for CCL2 blockade therapy. IHC measurement of CCR2 showed good correlation with MSCs and M2-like macrophages, supporting its biological role, and demonstrated higher relevance as a biomarker for this pathway than IHC measurement of CCL2. As a conclusion of this study, the following recommendations for CCL2 therapy can be summarized:

• In OC, CCL2-targeted therapy may be recommended due to the highest prevalence of CCL2 and CCR2 and M2 polarization;

• CCL2-targeted therapy may be recommended for PDAC due to the largest number of MSCs compared to other tumor types analyzed and because CCR2 and M2 were present at fairly high levels and numbers. CCL2 levels were also high in PDAC and, in contrast to other tumor types, it was demonstrated to be extraordinarily overrepresented in immune cells compared to tumor cells. PCa showed a very good correlation between CCL2/CCR2 and MSC recruitment/M2 polarization. These results suggest that in the analyzed PDAC samples, the role of CCL2-CCR2 is strongly focused on the recruitment of immune cells.

• In breast cancer, especially TNBC, CCL2-targeted therapy may be recommended: breast cancer demonstrated CCL2, CCR2, MSCs and M2 at fairly high levels and quantities. In particular, TNBC cases were characterized by higher numbers of M2 and MSCs than non-TNBC, although non-TNBC showed the highest production of CCL2 in tumor cells compared with other tumor indications.

The following tumor indications appeared to be less dependent on the CCL2-CCR2 axis and therefore for which CCL2-targeted therapy may be less recommended:

• CRC: CCL2 levels were low compared to other tumor types, especially compared to PCa. Also, the lowest number of M2-like macrophages was measured in this case compared to other tumor types. CCR2 and MSCs were present in variable amounts. Interestingly, only in this indication was there a trend towards a positive correlation between CCL2 and CCR2. However, the overall results suggest that in CRC the role of CCL2-CCR2 is focused on tumor cell survival rather than immune cell recruitment.

• Despite high levels of CCL2, low levels of CCR2 and the lowest number of MSCs were observed in EC. M2-like macrophages were present in variable numbers.

• In prostate cancer, CCR2 was present at variable levels. (M2 and MSC were not measured)

Correlation Analysis

The results of the study can be summarized as follows:

• A positive correlation was present between tumor CCL2 and CCR2 (IHC) only in CRC.

• Serum CCL2 (ELISA) does not correlate with any of the measured parameters in the tumor, including CCL2, CCR2, macrophages and MSCs. A trend towards a positive correlation with tumor CCL2 was found only in PCa, where both methods showed very low values.

• CCR2 expression correlates positively with the presence of M2-like macrophages and CD14 ⁺ cells and negatively with the M1/M2 ratio, and confirms the biological role of CCR2. CCR2 levels correlate better with M2 polarization than with MSC recruitment.

• CCL2 showed a tendency to be positively correlated with MSC recruitment and M2 polarization. Thus, the level of CCL2 alone does not appear to be a major factor for the presence of MSCs and M2-like polarization.

--->

LIST OF SEQUENCES

<110> F. Hoffmann-La Roche AG

<120> Bispecific anti-CCL2 antibodies

<130> P35823-WO

<150>EP19217665.9

<151> 2019-12-18

<160> 174

<170> Patent In version 3.5

<210> 1

<211> 3

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 1A4

<400> 1

Trp Ile Cys

1

<210> 2

<211> 18

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 1A4

<400> 2

Cys Ile Gly Ala Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

1 5 10 15

Lys Gly

<210> 3

<211> 10

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 1A4

<400> 3

Thr Gly Thr Glu Phe Thr Tyr Tyr Ser Leu

1 5 10

<210> 4

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 1A4

<400> 4

Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Met Ala

1 5 10

<210> 5

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 1A4

<400> 5

Thr Ala Ser Ser Leu Ala Ser

15

<210> 6

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 1A4

<400> 6

Ala Gly Tyr Lys Ser Tyr Ser Asn Asp Glu Tyr Gly

1 5 10

<210> 7

<211> 116

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 1A4

<400> 7

Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Glu Leu Asp Phe Tyr Trp Ile Cys

20 25 30

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala Cys Ile

35 40 45

Gly Ala Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly

50 55 60

Arg Phe Thr Val Ser Lys Thr Ser Ser Thr Thr Val Thr Leu Gln Met

65 70 75 80

Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Thr

85 90 95

Gly Thr Glu Phe Thr Tyr Tyr Ser Leu Trp Gly Pro Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 8

<211> 111

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL 1A4

<400> 8

Ala Leu Val Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Asn Asn

20 25 30

Asn Met Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Thr Ala Ser Ser Leu Ala Ser Gly Val Pro Ser His Phe Arg

50 55 60

Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu

65 70 75 80

Ser Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Tyr Lys Ser Tyr Ser

85 90 95

Asn Asp Glu Tyr Gly Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 9

<211> 6

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 1A5

<400> 9

Thr Ser Tyr Trp Met Cys

15

<210> 10

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 1A5

<400> 10

Cys Ile Ser Ser Ser Ile Gly Val Thr Tyr Tyr Ala Ser Trp Ala Glu

1 5 10 15

Gly

<210> 11

<211> 12

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 1A5

<400> 11

Thr Thr Asp Asp Asn Trp Asn Val Gly Phe Asn Leu

1 5 10

<210> 12

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 1A5

<400> 12

Gln Ala Ser Gln Ser Ile Gly Asn Arg Tyr Leu Ser

1 5 10

<210> 13

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 1A5

<400> 13

Gly Thr Ser Thr Leu Ala Ser

15

<210> 14

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 1A5

<400> 14

Gln Gln Gly Ala Thr Ile Ser Tyr Leu Asp Asn Ala

1 5 10

<210> 15

<211> 121

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 1A5

<400> 15

Gln Glu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser

20 25 30

Tyr Trp Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu

35 40 45

Ile Ala Cys Ile Ser Ser Ser Ile Gly Val Thr Tyr Tyr Ala Ser Trp

50 55 60

Ala Glu Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr

65 70 75 80

Leu Gln Met Thr Ser Leu Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Thr Thr Asp Asp Asn Trp Asn Val Gly Phe Asn Leu Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 16

<211> 111

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL 1A5

<400> 16

Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Gly Val Gly

1 5 10 15

Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Gly Asn Arg

20 25 30

Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys

50 55 60

Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu

65 70 75 80

Ser Ala Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Ile Ser

85 90 95

Tyr Leu Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 17

<211> 6

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 1G9

<400> 17

Leu Tyr Ser Tyr Met Cys

15

<210> 18

<211> 18

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 1G9

<400> 18

Cys Val Asp Ala Gly Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala

1 5 10 15

Lys Gly

<210> 19

<211> 19

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 1G9

<400> 19

Gly Ile Leu Tyr Tyr Thr Trp Pro Tyr Pro Ala Gly Ala Ile Asp Ala

1 5 10 15

Phe Asp Ser

<210> 20

<211> 11

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 1G9

<400> 20

Gln Ala Ser Glu Ser Ile Ser Asn Tyr Leu Ser

1 5 10

<210> 21

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 1G9

<400> 21

Lys Ala Ser Thr Leu Ala Ser

15

<210> 22

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 1G9

<400> 22

Gln Gln Ser Tyr Ser Ser Ser Asn Val Phe Asn Thr

1 5 10

<210> 23

<211> 128

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 1G9

<400> 23

Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Leu Thr Leu Thr Cys Lys Ala Ser Gly Ile Asp Phe Ser Leu Tyr Ser

20 25 30

Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Cys Val Asp Ala Gly Ala Ser Gly Ser Thr Tyr Tyr Ala Ser Trp

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr

65 70 75 80

Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Gly Ile Leu Tyr Tyr Thr Trp Pro Tyr Pro Ala Gly Ala Ile

100 105 110

Asp Ala Phe Asp Ser Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 110

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL 1G9

<400> 24

Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly

1 5 10 15

Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Glu Tyr Thr Val Thr Ile Ser Gly Val Gln Ser

65 70 75 80

Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Ser Asn

85 90 95

Val Phe Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 25

<211> 6

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 2F6

<400> 25

Asn Asn Tyr Tyr Met Cys

15

<210> 26

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 2F6

<400> 26

Cys Ile Ser Thr Asp Asp Ser Asn Thr His Tyr Ala Ser Trp Ala Gln

1 5 10 15

Gly

<210> 27

<211> 13

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 2F6

<400> 27

Asp Ala His Phe Thr Ser Tyr Gly Tyr Gly Phe Asp Leu

1 5 10

<210> 28

<211> 11

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 2F6

<400> 28

Arg Ala Ser Glu Asp Ile Glu Asn Leu Val Ala

1 5 10

<210> 29

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 2F6

<400> 29

Gln Ala Ser Lys Leu Ala Ser

15

<210> 30

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 2F6

<400> 30

Gln Gly Asp Tyr Gly Ser Gly Trp Ile Met Tyr Ser

1 5 10

<210> 31

<211> 121

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 2F6

<400> 31

Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly Ser

1 5 10 15

Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Asn Asn Asn Tyr

20 25 30

Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Ser Thr Asp Asp Ser Asn Thr His Tyr Ala Ser Trp Ala

50 55 60

Gln Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Ala Leu Thr Leu

65 70 75 80

Gln Val Ala Gly Leu Thr Val Ala Asp Met Ala Thr Tyr Phe Cys Ala

85 90 95

Arg Asp Ala His Phe Thr Ser Tyr Gly Tyr Gly Phe Asp Leu Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 110

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL 2F6

<400> 32

Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly

1 5 10 15

Gly Thr Val Ser Ile Asn Cys Arg Ala Ser Glu Asp Ile Glu Asn Leu

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Gly Asp Leu Glu Ser

65 70 75 80

Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Asp Tyr Gly Ser Gly Trp

85 90 95

Ile Met Tyr Ser Phe Gly Gly Gly Thr Asp Leu Val Val Lys

100 105 110

<210> 33

<211> 5

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 CNTO888

<400> 33

Ser Tyr Gly Ile Ser

15

<210> 34

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 CNTO888

<400> 34

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 35

<211> 10

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 CNTO888

<400> 35

Tyr Asp Gly Ile Tyr Gly Glu Leu Asp Phe

1 5 10

<210> 36

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 CNTO888

<400> 36

Arg Ala Ser Gln Ser Val Ser Asp Ala Tyr Leu Ala

1 5 10

<210> 37

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 CNTO888

<400> 37

Asp Ala Ser Ser Arg Ala Thr

15

<210> 38

<211> 10

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 CNTO888

<400> 38

His Gln Tyr Ile Gln Leu His Ser Phe Thr

1 5 10

<210> 39

<211> 119

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH CNTO888

<400> 39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Gly Ile Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 40

<211> 109

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL CNTO888

<400> 40

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile Gln Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 5

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 Humanized 11K2 (= 11K2)

<400> 41

Asp Thr Tyr Met His

15

<210> 42

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 Humanized 11K2 (= 11K2)

<400> 42

Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 43

<211> 8

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 Humanized 11K2 (= 11K2)

<400> 43

Gly Val Phe Gly Phe Phe Asp Tyr

15

<210> 44

<211> 11

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 Humanized 11K2 (= 11K2)

<400> 44

Lys Ala Thr Glu Asp Ile Tyr Asn Arg Leu Ala

1 5 10

<210> 45

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 Humanized 11K2 (= 11K2)

<400> 45

Gly Ala Thr Ser Leu Glu Thr

15

<210> 46

<211> 9

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 Humanized 11K2 (= 11K2)

<400> 46

Gln Gln Phe Trp Ser Ala Pro Tyr Thr

15

<210> 47

<211> 117

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH Humanized 11K2 (= 11K2)

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 48

<211> 107

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL Humanized 11K2 (= 11K2)

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 5

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 ABN912

<400> 49

His Tyr Trp Met Ser

15

<210> 50

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 ABN912

<400> 50

Asn Ile Glu Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210> 51

<211> 13

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 ABN912

<400> 51

Asp Leu Glu Gly Leu His Gly Asp Gly Tyr Phe Asp Leu

1 5 10

<210> 52

<211> 11

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 ABN912

<400> 52

Arg Ala Ser Gln Gly Val Ser Ser Ala Leu Ala

1 5 10

<210> 53

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 ABN912

<400> 53

Asp Ala Ser Ser Leu Glu Ser

15

<210> 54

<211> 9

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 ABN912

<400> 54

Gln Gln Phe Asn Ser Tyr Pro Leu Thr

15

<210> 55

<211> 122

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH ABN912

<400> 55

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Asn Ile Glu Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Asp Leu Glu Gly Leu His Gly Asp Gly Tyr Phe Asp Leu Trp

100 105 110

Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 56

<211> 107

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL ABN912

<400> 56

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ile Leu Thr Cys Arg Ala Ser Gln Gly Val Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Pro Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Phe Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 6

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 mutated variant CNTO888

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is I or T

<400> 57

Ser His Tyr Gly Xaa Ser

15

<210> 58

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 mutated variant CNTO888

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X is V or I or H

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is P or H

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X is H or G

<400> 58

Gly Xaa Ile Xaa Ile Phe Xaa Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 59

<211> 10

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 mutated variant CNTO888

<400> 59

Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe

1 5 10

<210> 60

<211> 12

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 mutated variant CNTO888

<400> 60

Arg Ala Ser Gln His Val Ser Asp Ala Tyr Leu Ala

1 5 10

<210> 61

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 mutated variant CNTO888

<400> 61

Asp Ala Ser Asp Arg Ala Glu

15

<210> 62

<211> 10

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 mutated variant CNTO888

<400> 62

His Gln Tyr Ile His Leu His Ser Phe Thr

1 5 10

<210> 63

<211> 29

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H1 mutated variant CNTO888

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe

20 25

<210> 64

<211> 14

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H2 mutated variant CNTO888

<400> 64

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 65

<211> 32

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H3 mutated variant CNTO888

<400> 65

Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 66

<211> 11

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H4 mutated variant CNTO888

<400> 66

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 67

<211> 23

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L1 mutated variant CNTO888

<400> 67

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210> 68

<211> 15

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L2 mutated variant CNTO888

<400> 68

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210> 69

<211> 32

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L3 mutated variant CNTO888

<400> 69

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210> 70

<211> 9

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L4 mutated variant CNTO888

<400> 70

Gly Gln Gly Thr Lys Val Glu Ile Lys

15

<210> 71

<211> 119

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant CNTO888 H0695

<400> 71

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 72

<211> 119

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant CNTO888 H0625

<400> 72

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile His Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 73

<211> 119

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant CNTO888 H0634

<400> 73

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Thr Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile His Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 74

<211> 119

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant CNTO888 H0635

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly His Ile His Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 75

<211> 109

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL mutated variant CNTO888 L0616

<400> 75

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 5

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H1 mutated variant humanized 11K2

<400> 76

His Thr Tyr Met His

15

<210> 77

<211> 17

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H2 mutated variant humanized 11K2

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is D or E

<400> 77

Arg Ile Asp Pro Xaa Asn His Asn Thr Lys Phe Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 78

<211> 8

<212>PRT

<213> Artificial

<220>

<223> heavy chain CDR-H3 mutated variant humanized 11K2

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X is D or E

<400> 78

Gly Val Phe Gly Phe Phe Xaa His

15

<210> 79

<211> 11

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L1 mutated variant humanized 11K2

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is F or T

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X is R or L

<400> 79

Lys Ala Xaa Glu Asp Ile Tyr Asn Arg Xaa Ala

1 5 10

<210> 80

<211> 7

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L2 mutated variant humanized 11K2

<400> 80

Gly Ala Thr Ser Leu Glu His

15

<210> 81

<211> 9

<212>PRT

<213> Artificial

<220>

<223> light chain CDR-L3 mutated variant humanized 11K2

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is W or R

<400> 81

Gln Gln Phe Xaa Ser Ala Pro Tyr Thr

15

<210> 82

<211> 30

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H1 mutated variant humanized 11K2

<400> 82

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser

20 25 30

<210> 83

<211> 14

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H2 mutated variant humanized 11K2

<400> 83

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 84

<211> 32

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H3 mutated variant humanized 11K2

<400> 84

Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 85

<211> 11

<212>PRT

<213> Artificial

<220>

<223> heavy chain FR-H4 mutated variant humanized 11K2

<400> 85

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 86

<211> 23

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L1 mutated variant 11K2

<400> 86

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210> 87

<211> 15

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L2 mutated variant humanized 11K2

<400> 87

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile His

1 5 10 15

<210> 88

<211> 32

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L3 mutated variant humanized 11K2

<400> 88

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 89

<211> 10

<212>PRT

<213> Artificial

<220>

<223> light chain FR-L4 mutated variant humanized 11K2

<400> 89

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 90

<211> 117

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant humanized 11K2

H1503

<400> 90

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 91

<211> 117

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant humanized 11K2

H1510

<400> 91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 92

<211> 117

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH mutated variant humanized 11K2

H1514

<400> 92

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Glu Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 93

<211> 107

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL mutated variant humanized 11K2

L1338

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 94

<211> 107

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL mutated variant humanized 11K2

L1201

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 95

<211> 107

<212>PRT

<213> homo sapiens

<400> 95

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 96

<211> 105

<212>PRT

<213> homo sapiens

<400> 96

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 97

<211> 328

<212>PRT

<213> homo sapiens

<400> 97

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 98

<211> 328

<212>PRT

<213> Artificial

<220>

<223> exemplary human heavy chain constant region derived from IgG1

with mutations L234A, L235A and P329G (Fcgamma receptor

silenced)

<400> 98

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 99

<211> 328

<212>PRT

<213> homo sapiens

<400> 99

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 100

<211> 328

<212>PRT

<213> Artificial

<220>

<223> exemplary human heavy chain constant region derived from IgG1

with mutations (SG105-IgG1 allotype - Fcgamma receptor

silenced)

<400> 100

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 101

<211> 328

<212>PRT

<213> Artificial

<220>

<223> SG1095-exemplary human heavy chain constant region derived

from

IgG1 including the mutations (Kabat EU numbering)

L235W/G236N/H268D/Q295L/A330K/K326T, Q311R/P343R, N434A,

Q438R/S440E

<400> 101

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Leu Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Thr Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 102

<211> 328

<212>PRT

<213> Artificial

<220>

<223> SG1099-exemplary human heavy chain constant region derived

from

IgG1 including mutations (Kabat EU numbering) Q311R/P343R

<400> 102

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 103

<211> 328

<212>PRT

<213> Artificial

<220>

<223> SG1100-exemplary human heavy chain constant region derived

from

IgG1 including the mutations (Kabat EU numbering) Q311R/P343R,

N434A, Q438R/S440E

<400> 103

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 104

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CNTO888//11K2-WT IgG1

<400> 104

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 105

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CNTO888//11K2-WT IgG1

<400> 105

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Gly Ile Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 106

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CNTO888//11K2-WT IgG1

<400> 106

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 107

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CNTO888//11K2-WT IgG1

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile Gln Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 108

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 IgG1

<400> 108

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 109

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 IgG1

<400> 109

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 110

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 IgG1

<400> 110

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 111

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 IgG1

<400> 111

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 112

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - SG1095

<400> 112

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 113

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - SG1095

<400> 113

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Trp Asn Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 114

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - SG1095

<400> 114

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 115

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - SG1095

<400> 115

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 116

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - SG1099

<400> 116

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 117

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - SG1099

<400> 117

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 118

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - SG1099

<400> 118

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 119

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - SG1099

<400> 119

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 120

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - SG1100

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 121

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - SG1100

<400> 121

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 122

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - SG1100

<400> 122

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 123

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - SG1100

<400> 123

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 124

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO3 - SG1095

<400> 124

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Trp Asn Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 125

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO3 - SG1095

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Trp Asn Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Asp Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Leu Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Thr Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 126

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO3 - SG1095

<400> 126

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 127

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO3 - SG1095

<400> 127

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 128

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO3 - SG1099

<400> 128

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 129

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO3 - SG1099

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 130

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO3 - SG1099

<400> 130

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 131

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO3 - SG1099

<400> 131

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 132

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO3 - SG1100

<400> 132

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 133

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO3 - SG1100

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 134

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO3- SG1100

<400> 134

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 135

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO3 - SG1100

<400> 135

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 136

<211> 115

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 2F2

<400> 136

Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Thr Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Ser Phe Ile Asp His

20 25 30

Tyr Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Tyr Ile Gly Gly Ser Gly Thr Thr Tyr Tyr Ala Asn Arg Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Thr Ser Ser Thr Thr Val Thr Leu Gln

65 70 75 80

Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg

85 90 95

Asn Leu Asp Val Ser Ala Ser Leu Trp Gly Pro Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 137

<211> 112

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL 2F2

<400> 137

Gln Val Leu Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly Gly

1 5 10 15

Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn Asn

20 25 30

Leu Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Pro Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Gln Ser Thr Leu Thr Ile Ser Gly Val Gln

65 70 75 80

Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Glu Gly Ala Phe Leu Cys Thr

85 90 95

Thr Tyr Asp Cys Phe Val Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 138

<211> 117

<212>PRT

<213> mus musculus

<400> 138

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Ala Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Pro Ala Ser Gly Leu Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 139

<211> 107

<212>PRT

<213> mus musculus

<400> 139

Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Arg Leu Leu Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 140

<211> 121

<212>PRT

<213> Artificial

<220>

<223> heavy chain variable domain VH 1H11

<400> 140

Gln Glu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Glu Gly

1 5 10 15

Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Thr Ser

20 25 30

Tyr Trp Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu

35 40 45

Ile Ala Cys Ile Ser Ser Ser Ile Gly Val Thr Tyr Tyr Ala Ser Trp

50 55 60

Ala Glu Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Thr

65 70 75 80

Leu Gln Met Thr Ser Leu Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Thr Thr Asp Asp Asn Trp Asn Val Gly Phe Asn Leu Trp Gly

100 105 110

Pro Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 141

<211> 110

<212>PRT

<213> Artificial

<220>

<223> light chain variable domain VL 1H11

<400> 141

Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Gly Val Gly Gly

1 5 10 15

Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Gly Asn Arg Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys

65 70 75 80

Ala Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Ile Ser Tyr

85 90 95

Leu Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105 110

<210> 142

<211> 76

<212>PRT

<213> homo sapiens

<400> 142

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210> 143

<211> 76

<212>PRT

<213> Artificial

<220>

<223> exemplary human CCL2 (MCP1) - P8A variant

<400> 143

Gln Pro Asp Ala Ile Asn Ala Ala Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210> 144

<211> 76

<212>PRT

<213> Artificial

<220>

<223> exemplary human CCL2 (MCP1) - T10C variant

<400> 144

Gln Pro Asp Ala Ile Asn Ala Pro Val Cys Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr

65 70 75

<210> 145

<211> 76

<212>PRT

<213> homo sapiens

<400> 145

Gln Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile

1 5 10 15

Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr

20 25 30

Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly

35 40 45

Lys Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met

50 55 60

Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro

65 70 75

<210> 146

<211> 76

<212>PRT

<213> Artificial

<220>

<223> SEQ ID NO: 146 exemplary human CCL8 (MCP2) - P8A variant

<400> 146

Gln Pro Asp Ser Val Ser Ile Ala Ile Thr Cys Cys Phe Asn Val Ile

1 5 10 15

Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr

20 25 30

Asn Ile Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly

35 40 45

Lys Glu Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met

50 55 60

Lys His Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro

65 70 75

<210> 147

<211> 76

<212>PRT

<213> homo sapiens

<400> 147

Gln Pro Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe Ile

1 5 10 15

Asn Lys Lys Ile Pro Lys Gln Arg Leu Glu Ser Tyr Arg Arg Thr Thr

20 25 30

Ser Ser His Cys Pro Arg Glu Ala Val Ile Phe Lys Thr Lys Leu Asp

35 40 45

Lys Glu Ile Cys Ala Asp Pro Thr Gln Lys Trp Val Gln Asp Phe Met

50 55 60

Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys Leu

65 70 75

<210> 148

<211> 82

<212>PRT

<213> homo sapiens

<400> 148

Phe Asn Pro Gln Gly Leu Ala Gln Pro Asp Ala Leu Asn Val Pro Ser

1 5 10 15

Thr Cys Cys Phe Thr Phe Ser Ser Lys Lys Ile Ser Leu Gln Arg Leu

20 25 30

Lys Ser Tyr Val Ile Thr Thr Ser Arg Cys Pro Gln Lys Ala Val Ile

35 40 45

Phe Arg Thr Lys Leu Gly Lys Glu Ile Cys Ala Asp Pro Lys Glu Lys

50 55 60

Trp Val Gln Asn Tyr Met Lys His Leu Gly Arg Lys Ala His Thr Leu

65 70 75 80

Lys Thr

<210> 149

<211> 76

<212>PRT

<213> macaca fascicularis

<400> 149

Gln Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr

1 5 10 15

Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr

20 25 30

Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala

35 40 45

Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met

50 55 60

Asp His Leu Asp Lys Gln Ile Gln Thr Pro Lys Pro

65 70 75

<210> 150

<211> 125

<212>PRT

<213> mus musculus

<400> 150

Gln Pro Asp Ala Val Asn Ala Pro Leu Thr Cys Cys Tyr Ser Phe Thr

1 5 10 15

Ser Lys Met Ile Pro Met Ser Arg Leu Glu Ser Tyr Lys Arg Ile Thr

20 25 30

Ser Ser Arg Cys Pro Lys Glu Ala Val Val Phe Val Thr Lys Leu Lys

35 40 45

Arg Glu Val Cys Ala Asp Pro Lys Lys Glu Trp Val Gln Thr Tyr Ile

50 55 60

Lys Asn Leu Asp Arg Asn Gln Met Arg Ser Glu Pro Thr Thr Leu Phe

65 70 75 80

Lys Thr Ala Ser Ala Leu Arg Ser Ser Ala Pro Leu Asn Val Lys Leu

85 90 95

Thr Arg Lys Ser Glu Ala Asn Ala Ser Thr Thr Phe Ser Thr Thr Thr

100 105 110

Ser Ser Thr Ser Val Gly Val Thr Ser Val Thr Val Asn

115 120 125

<210> 151

<211> 328

<212>PRT

<213> Artificial

<220>

<223> GG01 - exemplary human heavy chain constant region derived

from

IgG1 including the mutations (Kabat EU numbering)

L234Y/P238D/T250V/V264I/T307P/A330K, Q311R/P343R, N434A,

Q438R/S440E

<400> 151

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 152

<211> 328

<212>PRT

<213> Artificial

<220>

<223> GG02 - exemplary human heavy chain constant region derived

from

IgG1 including mutations (Kabat EU numbering)

L234Y/P238D/T250V/V264I/T307P/A330K, Q311R/P343R,

M428L/N434A/Y436T,Q438R/S440E

<400> 152

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 153

<211> 328

<212>PRT

<213> Artificial

<220>

<223> GG03 -exemplary human heavy chain constant region derived from

IgG1 (compressing-IgG1 allotype sequences) including the

mutations

(Kabat EU numbering) L234Y/P238D/T250V/V264I/T307P/A330K,

Q311R/P343R,N434A,Q438R/S440E

<400> 153

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 154

<211> 328

<212>PRT

<213> Artificial

<220>

<223> GG04 -exemplary human heavy chain constant region derived from

IgG1 (compressing-IgG1 allotype sequences) including mutations

(Kabat EU numbering) L234Y/P238D/T250V/V264I/T307P/A330K,

Q311R/P343R, M428L/N434A/Y436T, Q438R/S440E

<400> 154

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Ile Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu

180 185 190

His Arg Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Arg Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr

305 310 315 320

Arg Lys Glu Leu Ser Leu Ser Pro

325

<210> 155

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - GG01

<400> 155

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 156

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - GG01

<400> 156

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 157

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 GG01

<400> 157

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 158

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 GG01

<400> 158

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 159

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 GG02

<400> 159

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 160

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 GG02

<400> 160

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 161

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - GG02

<400> 161

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 162

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - GG02

<400> 162

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 163

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - GG03

<400> 163

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 164

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - GG03

<400> 164

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 165

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - GG03

<400> 165

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 166

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - GG03

<400> 166

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 167

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - GG04

<400> 167

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 168

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - GG04

<400> 168

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 169

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - GG04

<400> 169

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 170

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - GG04

<400> 170

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 171

<211> 437

<212>PRT

<213> Artificial

<220>

<223> heavy chain 1- CKLO2 - GG03/GG04

<400> 171

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Phe Glu Asp Ile Tyr Asn Arg

20 25 30

Arg Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Gly Ala Thr Ser Leu Glu His Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Tyr Leu Gly Gly Asp Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Val Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Ile

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Val Leu His Arg Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Pro Lys Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu

420 425 430

Leu Ser Leu Ser Pro

435

<210> 172

<211> 447

<212>PRT

<213> Artificial

<220>

<223> heavy chain 2- CKLO2 - GG03/GG04

<400> 172

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser His Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Phe His Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Ala His Tyr Gly Glu Leu Asp Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Tyr Leu Gly Gly Asp

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Val Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Ile Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Pro Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Lys Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

420 425 430

Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

435 440 445

<210> 173

<211> 224

<212>PRT

<213> Artificial

<220>

<223> light chain 1- CKLO2 - GG03/GG04

<400> 173

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Leu Thr Ile Ser His Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asp Asn His Asn Thr Lys Phe Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Phe Gly Phe Phe Glu His Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 174

<211> 216

<212>PRT

<213> Artificial

<220>

<223> light chain 2- CKLO2 - GG03/GG04

<400> 174

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Ser Asp Ala

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Asp Arg Ala Glu Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Ile His Leu His

85 90 95

Ser Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<---

Claims (28)

1. A bispecific antibody that specifically binds to human CCL2, comprising a first antigen binding site that binds to a first epitope on human CCL2 and a second distinct antigen binding site that binds to a second distinct epitope on human CCL2, wherein the bispecific antibody contains an Fc domain human IgG isotype, where i) said first antigen binding site comprises a VH domain containing (a) a CDR-H1 containing the amino acid sequence SHYGXS of SEQ ID NO: 57, where X is I, (b) a CDR-H2 containing the amino acid sequence GX¹IX²IFX³TANYAQKFQG from SEQ ID NO: 58, where X¹ represents V, X² represents P and X³ represents H, and (c) CDR-H3 containing the amino acid sequence YDAHYGELDF from SEQ ID NO: 59; And a VL domain containing (d) CDR-L1 containing the amino acid sequence RASQHVSDAYLA from SEQ ID NO: 60, (e) CDR-L2 containing the amino acid sequence DASDRAE from SEQ ID NO: 61, and (f) CDR-L3 containing the amino acid sequence HQYIHLHSFT from SEQ ID NO: 62; And ii) said second antigen binding site comprises a VH domain comprising (a) a CDR-H1 containing the amino acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 containing the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77, where X represents is D, and (c) CDR-H3 containing the amino acid sequence GVFGFFXH from SEQ ID NO: 78, where X is E; And VL domain containing (d) CDR-L1 containing the amino acid sequence KAX¹EDIYNRX²A from SEQ ID NO: 79 where X¹ represents F and X² is R, (e) CDR-L2 containing the amino acid sequence GATSLEH from SEQ ID NO: 80, and (f) CDR-L3 containing the amino acid sequence QQFXSAPYT from SEQ ID NO: 81, where X is W. 2. Bispecific antibody according to claim 1, where i) said first antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 71; and a VL domain containing the amino acid sequence of SEQ ID NO: 75; And ii) said second antigen binding site comprises a VH domain containing the amino acid sequence of SEQ ID NO: 91; and a VL domain containing the amino acid sequence of SEQ ID NO: 93. 3. Bispecific antibody according to any one of paragraphs. 1, 2, wherein the bispecific antibody containing the Fc domain of the human IgG isotype is a bispecific antibody containing the heavy chain constant domain of the human IgG1 isotype. 4. The bispecific antibody according to any of the preceding claims, wherein the bispecific antibody binds to human CCL2 with 10 times greater affinity at pH 7.4 than at pH 5.8. 5. A bispecific antibody according to any of the preceding claims, wherein the bispecific antibody comprises a human IgG1 heavy chain constant domain containing one or more of the following mutations (Kabat EU numbering): i) Q311R and/or P343R; and/or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and/or A330K; and/or iii) M428L, N434A and/or Y436T; and/or iv) Q438R and/or S440E. 6. An isolated nucleic acid encoding an antibody according to any of the previous paragraphs. 7. A host cell that produces a bispecific antibody according to any one of claims 1-5, containing the isolated nucleic acid according to claim 6. 8. A method for producing an antibody, comprising culturing a host cell according to claim 7 to obtain the antibody. 9. The method of claim 8, further comprising isolating the antibody from the host cell. 10. Pharmaceutical composition for use in the treatment of cancer, containing a bispecific antibody according to any one of paragraphs. 1-5 and a pharmaceutically acceptable carrier. 11. Pharmaceutical composition for use in the treatment of an inflammatory disease, containing a bispecific antibody according to any one of paragraphs. 1-5 and a pharmaceutically acceptable carrier. 12. Pharmaceutical composition for use in the treatment of an autoimmune disease, containing a bispecific antibody according to any one of paragraphs. 1-5 and a pharmaceutically acceptable carrier. 13. Use of a bispecific antibody according to any one of paragraphs. 1-5 as a drug in the treatment of cancer. 14. Use of a bispecific antibody according to any one of paragraphs. 1-5 as a drug in the treatment of an inflammatory disease. 15. Use of a bispecific antibody according to any one of paragraphs. 1-5 as a drug in the treatment of an autoimmune disease. 16. Use of a bispecific antibody according to any one of paragraphs. 1-5 in cancer treatment. 17. Use of a bispecific antibody according to any one of paragraphs. 1-5 in the treatment of inflammatory disease. 18. Use of a bispecific antibody according to any one of paragraphs. 1-5 in the treatment of autoimmune disease.