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CN117295762A - anti-CD 122 antibodies and uses thereof - Google Patents

anti-CD 122 antibodies and uses thereof Download PDF

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Publication number
CN117295762A
CN117295762A CN202280028157.8A CN202280028157A CN117295762A CN 117295762 A CN117295762 A CN 117295762A CN 202280028157 A CN202280028157 A CN 202280028157A CN 117295762 A CN117295762 A CN 117295762A
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antibody
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W·J·J·芬利
J·E·哈里斯
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Villaris Treatment Co ltd
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Villaris Treatment Co ltd
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Priority claimed from PCT/US2022/024620 external-priority patent/WO2022221409A1/en
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Abstract

Provided herein are antibody molecules and antigen-binding portions thereof that specifically bind CD122, and related compositions, nucleic acid molecules, vectors, and host cells. Also provided herein are medical uses for such antibody molecules.

Description

anti-CD 122 antibodies and uses thereof
Cross Reference to Related Applications
The present application claims priority from U.S. provisional patent application Ser. No. 63/279,762, filed on 11/16/2021, and U.S. provisional patent application Ser. No. 63/174,772, filed on 4/2021, each of which is incorporated herein by reference in its entirety for all purposes.
Description of electronically submitted text files
The contents of the text file submitted electronically herewith are incorporated herein by reference in its entirety: a computer-readable format copy of the sequence listing (filename: vlrs_001_02wo_seqlist_st25.Txt, date of record: 2022, month 4, day 13, file size about 87,964 bytes).
Technical Field
The present disclosure relates to therapeutic antibody molecules and medical uses thereof.
Background
CD122 is a cell surface receptor, a member of the immunoglobulin superfamily, and is expressed primarily on natural killer cells (NK) and T cells. CD122 has been proposed as a target for a variety of disorders driven by any of these immune cell types, including type 1 diabetes (T1D), celiac disease, leukemia, vitiligo, and the like. There is a need for therapeutic methods for such immune-mediated diseases. In particular, vitiligo has no systemic treatment options, nor has there been a medical treatment approved by the U.S. food and drug administration (U.S. food and Drug Administration) to ameliorate disease.
Disclosure of Invention
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO:7 and the VL region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO:15, or (b) the VH region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO:7 and the VL region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises SEQ ID NO:1 and the VL region amino acid sequence comprises SEQ ID NO:17; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 and the VL region amino acid sequence comprises SEQ ID NO:9.
in some embodiments, the antibody or antigen binding portion is humanized or chimeric.
In some embodiments, the VH region, the VL region, or both the VH region and the VL region comprise one or more human framework region amino acid sequences. In some embodiments, the VH region, the VL region, or both the VH region and the VL region comprise a human variable region framework amino acid sequence into which CDR amino acid sequences have been inserted. In some embodiments, the VH region comprises an IGHV3-23 human germline scaffold amino acid sequence into which HCDR1, HCDR2, and HCDR3 amino acid sequences have been inserted. In some embodiments, the VL region comprises IGKV1-33 human germline scaffold amino acid sequences into which LCDR1, LCDR2, and LCDR3 amino acid sequences have been inserted.
In some embodiments, the anti-CD 122 antibody comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, igE, igM, igD, igA or IgY. In some embodiments, the immunoglobulin constant region is IgG1, igG2, igG3, igG4, igA1, or IgA2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitutions S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A, L a and G237A, or a wild-type human IgG2 constant region, wherein the numbering is according to the EU index as in Kabat. In some embodiments, the immunoglobulin constant region comprises SEQ ID NO: 32-38.
In some embodiments, the antibody or antigen binding portion is a Fab, fab ', F (ab') 2 Fv, scFv, macroantibody, minibody, diabody, triabody, tetrabody or diabody. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody is a tetrameric antibody, a tetravalent antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody that specifically binds a first antigen and a second antigen, wherein the first antigen is CD122 and the second antigen is not CD122.
Provided herein is an immunoconjugate comprising an antibody or antigen binding portion disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, radioisotope, chemotherapeutic agent, immunomodulatory agent, cytostatic enzyme, cytolytic enzyme, therapeutic nucleic acid, anti-angiogenic agent, anti-proliferative agent, or pro-apoptotic agent.
Provided herein is a pharmaceutical composition comprising an antibody, antigen-binding portion or immunoconjugate disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient.
Provided herein is a nucleic acid molecule encoding the amino acid sequence of the (a) VH region of an antibody or antigen-binding portion disclosed herein; (b) VL region amino acid sequence; or (c) both the VH region amino acid sequence and the VL region amino acid sequence. Provided herein is an expression vector comprising a nucleic acid molecule disclosed herein. Provided herein is a recombinant host cell comprising a nucleic acid molecule or expression vector disclosed herein.
Provided herein is a method of producing an anti-CD 122 antibody, or antigen-binding portion thereof, comprising: culturing a recombinant host cell comprising an expression vector disclosed herein under conditions that express the nucleic acid molecule, thereby producing the antibody or antigen-binding portion; and isolating the antibody or antigen binding portion from the host cell or culture.
Provided herein is a method for inhibiting an immune response in a subject comprising administering to the subject a therapeutically effective amount of an antibody, antigen binding portion, immunoconjugate or pharmaceutical composition disclosed herein. In some embodiments, the immune response is mediated by CD 122.
Provided herein is a method for treating or preventing a disease in a subject comprising administering to the subject a therapeutically effective amount of an antibody, antigen-binding portion, immunoconjugate, or pharmaceutical composition disclosed herein. In some embodiments, the disease is an inflammatory disease or an autoimmune disease. In some embodiments, the disease is vitiligo, celiac disease, type 1 diabetes, multiple sclerosis, graft versus host disease, systemic lupus erythematosus, psoriasis, atopic dermatitis, alopecia areata, ulcerative colitis, or rheumatoid arthritis.
Provided herein is a method for inhibiting IL-15-induced T cell migration from skin comprising contacting the skin with a therapeutically effective amount of an antibody, antigen binding portion, immunoconjugate or pharmaceutical composition disclosed herein.
Provided herein are antibodies, antigen binding portions, immunoconjugates, or pharmaceutical compositions disclosed herein for use as a medicament.
Drawings
Figure 1 depicts a graph of in vivo pharmacological analysis of anti-CD 122 IgG.
FIGS. 2A-2D depict data from a specific assay for Villmab-1 (MIK. Beta.1) anti-CD 122 IgG. The proteome-specific profile of Villmab 1 was constructed using Retrogenix technology. (FIG. 2A) ZS control expression, (FIG. 2B) Villmab-1 probe, (FIG. 2C) rituximab probe, (FIG. 2D) no primary antibody.
Figures 3A-3C depict data from flow cytometry analysis of Villmab-1 (mikβ1) anti-CD 122 IgG on target transfected cells. (FIG. 3A) Villmab-1 binding, (FIG. 3B) no primary antibody, (FIG. 3C) rituximab binding.
Figure 4 depicts data for anti-CD 122 IgG Alphascreen epitope competition. Novel clones were screened in the Alphascreen assay to check for competition with the Villmab-1 binding epitope on human CD 122.
Fig. 5 depicts a bar graph showing the multiple reactivity scores of novel anti-CD 122 IgG. Antibodies were examined for their ability to bind non-specifically to DNA and insulin.
FIGS. 6A-6G depict data from flow cytometry analysis of novel anti-CD 122 IgG and Villmab-1 on target transfected cells. (fig. 6A) 06F11, (fig. 6B) 07C07, (fig. 6C) 07D06, (fig. 6D) 07E09, (fig. 6E) 07D07, (fig. 6F) 06D12, (fig. 6G) isotype control IgG.
FIGS. 7A-7F depict data from analysis of novel anti-CD 122 IgG and Villmab-1 in an M07e cell IL-15 proliferation assay. (fig. 7A) 06F11, (fig. 7B) 07C07, (fig. 7C) 07D06, (fig. 7D) 07E09, (fig. 7E) 07D07, (fig. 7F) 06D12.
FIGS. 8A-8F depict data from in vivo analysis of novel anti-CD 122 IgG and Villmab-1 in a hIL-15 NSG mouse model. (FIG. 8A) previous human CD8+ T cells, (FIG. 8B) previous human NK cells, (FIG. 8C) human CD8+ T cells after 1 week, (FIG. 8D) human NK cells after 1 week, (FIG. 8E) human CD8+ T cells after 3 weeks, (FIG. 8F) human NK cells after 3 weeks.
FIGS. 9A-9D depict data from flow cytometry analysis of novel anti-CD 122 IgG and Villmab-1 on target transfected cells. (fig. 9A) 06F11, (fig. 9B) 07C07, (fig. 9C) isotype control IgG, (fig. 9D) rituximab.
FIGS. 10A-10C depict data from specific analysis of novel clones of Villmab-1 (MIK. Beta.1) anti-CD 122 in Fab and IgG forms. UsingThe technique measures Fab-specific profiles against the human neudins protein (fig. 10A) or the CILP2 protein (fig. 10B) as Rmax (maximum specific binding response value). The specificity profile of IgG against human BCAM protein (fig. 10C) was measured as OD450nm using ELISA.
FIGS. 11A-11B depict data from sequence analysis of Villmab-1 (MIK.beta.1) anti-CD 122 novel cloned variable domain sequences. VH sequences (fig. 11A) and VL sequences (fig. 11B). In fig. 11A, the sequence is as follows: villMAB-1: SEQ ID NO:22; MAB05: SEQ ID NO:1, a step of; MAB06: SEQ ID NO:1, a step of; MAB14: SEQ ID NO:52; MAB15: SEQ ID NO:52; MAB17: SEQ ID NO:53; MAB18: SEQ ID NO:53. in fig. 11B, the sequence is as follows: villMAB-1: SEQ ID NO:28; MAB05: SEQ ID NO:9, a step of performing the process; MAB06: SEQ ID NO:17; MAB14: SEQ ID NO:9, a step of performing the process; MAB15: SEQ ID NO:17; MAB17: SEQ ID NO:9, a step of performing the process; MAB18: SEQ ID NO:17. CDRs are bold and underlined. Residues other than the VillMab-1 sequence are highlighted in grey boxes. Only the unique residues found in the clones in this analysis that do not bind BCAM, CILP2 or neudins are marked with black boxes.
FIGS. 12A-12B depict data from analysis of novel anti-CD 122 IgG and Villmab-1 in a primary NK cell IL-15 proliferation assay. MAB05 (FIG. 12A), MAB06 (FIG. 12B).
Fig. 13A-13B depict analytical data for the effect of MAB05 and MAB06 on IL 15-induced T cell accumulation of migration from skin biopsies in a human skin biopsy culture assay. Figure 13A shows the effect on cd8+ T cell number. Figure 13B shows the effect on cd4+ T cell numbers.
FIGS. 14A-14B depict data from an analysis of IL 15-induced concentration-dependent antagonism of MAB05 and MAB06 for T cell accumulation from skin biopsy migration in a human skin biopsy culture assay. Figure 13A shows the effect on cd8+ T cells and associated IC 50. Fig. 13B shows the effect on cd4+ T cells and related IC 50.
Detailed Description
Provided herein are anti-CD 122 antibodies and therapeutic uses of such antibodies. The antibodies disclosed herein are antagonistic, well expressed, biophysically stable, highly soluble and have maximized identity to the preferred human germline.
CD122 (also known as IL2RB, IL-2 Rbeta, IL15RB, P70-75, interleukin 2 receptor subunit beta, and IMD 63) is a type I transmembrane glycoprotein and is a member of the Ig superfamily. CD122 is a common subunit of the interleukin-15 (IL-15) receptor and the interleukin-2 (IL-2) receptor. CD122 is expressed by NK cells and T cell subsets. IL-15 signaling is associated with the pathogenesis of human vitiligo. Targeting CD122 or blocking IL-15 signaling appears to be beneficial in other mouse models of immune-mediated diseases such as diabetes, psoriasis, multiple sclerosis, and alopecia areata, as well as in ameliorating the symptoms of rheumatoid arthritis and celiac disease. Development of potent antagonistic CD122 antibodies would be valuable for the treatment of immune-mediated diseases.
U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety, describes the preparation of an antagonistic murine anti-CD 122 IgG molecule, known as "MIKβ1", and a humanized form of MIKβ1. These humanized forms of MIKβ1 were generated using classical humanization techniques, i.e., by grafting Kabat-defined murine CDRs into human heavy and light chain framework sequences, some of which were potentially back mutated to correspondingly located MIKβ1 murine residues. The partially humanized form of MIKβ1 (see Table 20) showed no efficacy in phase IIa clinical trials against T cell large granular lymphocyte (T-LGL) leukemia and human T cell lymphotropic virus type 1 (HTLV-1) associated myelopathy/Tropical spastic paresis (HAM/TSP). This antibody has a number of disadvantages including the fact that: it is partially humanized with off-target binding that can affect pharmacokinetics and biodistribution, and uses IgG1 isotypes, resulting in the risk of unnecessary antibody-dependent cytotoxicity/antibody-dependent cellular phagocytosis on cells that do not mediate the disease. These features make this antibody a suboptimal candidate for further testing as a targeted therapy for human immune-mediated diseases.
In contrast, the anti-CD 122 antibodies provided herein exhibit advantages as described herein, making them useful for the treatment of human immune-mediated diseases and disorders.
Antibodies to
Provided herein are antibodies and antigen binding portions thereof that specifically bind CD122. The anti-CD 122 antibodies provided herein have several advantages over the murine anti-CD 122 antibody mikβ1 disclosed in US 5,585,089 and humanized versions thereof. The anti-CD 122 antibodies provided herein have been selected to have increased efficacy in blocking IL-15 signaling by CD122. Importantly, these antibodies also significantly improved the specificity of CD122 binding by eliminating off-target binding to the human receptors BCAM (also known as AU, CD239, LU, MSK19, basal cell adhesion molecule (Lutheran blood group)), neudins (also known as NENF) and CILP2 (also known as cartilage intermediate layer protein 2) compared to mikβ1.
The antibodies and antigen binding portions disclosed herein specifically bind to human CD122. In some embodiments, the antibodies and antigen binding portions may cross-react with CD122 from a species other than human, such as cynomolgus monkey (Macaca fascicularis)) CD122 and/or rhesus monkey (Macaca mulatta) CD122. In some embodiments, the antibody may be specific for human CD122 alone, and may not exhibit non-human cross-reactivity. Table 16 provides exemplary amino acid sequences for human CD122, cynomolgus monkey CD122 and rhesus monkey CD122.
The term "antibody" broadly refers to an immunoglobulin (Ig) molecule that typically comprises four polypeptide chains, i.e., two heavy chains (H) and two light chains (L), or any functional fragment, mutant, variant, or derivative thereof that retains the basic target binding characteristics of an Ig molecule. Such mutant, variant or derived antibody forms are known in the art.
In full length antibodies, each heavy chain comprises a heavy chain variable region (abbreviated herein as VH region) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL region) and a light chain constant region. The light chain constant region comprises one domain, i.e., CL. VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH domain and VL domain consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDR definitions as used in this application are Kabat definitions (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition Bethesda, MD: public Health Service, national Institutes of Health (1991)).
The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as the stretch from position Cys226 or from the amino acid residue of Pro230 to its carboxy-terminus. Numbering of residues in the Fc region is according to the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. The Fc region may exist as a dimer or monomer. The Fc region binds to various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.
Immunoglobulin molecules may be of any type (e.g., coffee IgG, igE, igM, igD, igA or IgY) and class (e.g., coffee IgG1, igG2, igG3, igG4, igA1 or IgA 2) or subclass. IgG, igD and IgE antibodies typically comprise two identical heavy chains and two identical light chains and two antigen-binding domains, each domain consisting of one VH and one VL. IgA antibodies typically consist of two monomers, each consisting of two heavy and two light chains (as IgG, igD and IgE antibodies); in this way, the IgA molecule has four antigen binding domains, each consisting in turn of VH and VL. Some IgA antibodies are monomeric in that they consist of two heavy and two light chains. Secretory IgM antibodies are generally composed of five monomers, each consisting of two heavy and two light chains (as are IgG and IgE antibodies). Thus, an IgM molecule has ten antigen binding domains, each domain in turn consisting of VH and VL. The cell surface form of IgM has two heavy/two light chain structures similar to IgG, igD and IgE antibodies.
As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody (or "antibody portion" or "antibody fragment") refers to one or more antibody fragments that retain the ability to specifically bind an antigen (e.g., CD 122). It has been shown that the antigen binding function of an antibody can be achieved by a portion or fragment of a full length antibody. Examples of binding moieties included within the term "antigen-binding portion" of an antibody include (i) monovalent fragment Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Bivalent fragment F (ab') comprising two Fab fragments linked at the hinge region by a disulfide bridge 2 Fragments; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb (domain antibody) fragments comprising a single variable domain (Ward et al, (1989) Nature 341:544-546; WO 90/05144 A1, each of which is incorporated herein by reference in its entirety); and (vi) an isolated Complementarity Determining Region (CDR). The disclosure also includes Fab' fragments. Fab 'fragments can be purified by reduction of F (ab') 2 Fragments are formed. Fab 'is derived from F (ab') 2 The method comprises the steps of carrying out a first treatment on the surface of the Thus, it may contain a small portion of Fc. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that makes them a single protein chain, in which the VL domain and VH domain pair to form monovalent molecules, known as single chain Fv (scFv). See, e.g., bird et al (1988) Science 242:423-426; huston et al (1988) Proc.Natl. Acad. Sci.USA 85:5879-5883. Such single chain antibodies are also intended to be encompassed by the term "antigen binding portion" of an antibody. In some embodiments In this case, the scFv molecule may be incorporated into a fusion protein. In some embodiments, provided herein are single chain camelidae antibodies. In some embodiments, provided herein are shark heavy chain antibodies (V-NARs). See englist et al (2020) Antibody Therapeutics,3 (1): 1-9. Examples of antigen binding moieties are known in the art (Kontermann and Dubel et al, antibody Engineering (2001) Springer-Verlag. New York. Page 790). In some embodiments, provided herein is a single domain antibody. Generally, the term "antibody" as used herein includes "antibody portions". The antibody portion generally retains the antigen binding properties of the full length antibody.
Antibodies and antibody portions provided herein can be in a multispecific (e.g., coffee, bispecific or trispecific) form. Such multispecific molecules specifically bind to two or more different molecular targets or epitopes. In some embodiments, the antibody or antigen binding portion is a bispecific molecule that specifically binds a first antigen and a second antigen, wherein the first antigen is CD122 and the second antigen is not CD122. In some embodiments, the antibody or antigen binding portion is a diabody. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to pair between two domains on the same chain, forcing the domains to pair with the complementary domain of the other chain and creating two antigen binding sites (see e.g. coffee Holliger et al (1993) proc. Natl. Acad. Sci. USA 90:6444-6448; poljak et al (1994) Structure 2:1121-1123). In some embodiments, the antibody or antigen binding portion is a diabody, a tetrabody, a diabody, or a tandem scFv. In some embodiments, the antibody or antigen binding portion is a dual affinity retargeting protein.
In some embodiments, the anti-CD 122 antigen binding portions disclosed herein are Fab, fab ', F (ab') 2 Fv, scFv, macroantibody, minibody, diabody, triabody, tetrabody or diabody.
The terms "immunobinding" and "immunobinding properties" as used herein refer to immunoglobulin molecules (e.g., antibodies or antigens thereofBinding moiety) and the antigen to which the immunoglobulin is specific. The intensity or affinity of the immunological binding interactions can be determined by the dissociation constant (K d ) Wherein a smaller Kd indicates a greater affinity. The immunological binding properties of the selected polypeptides may be quantified using methods well known in the art. One such method entails measuring the rate of antigen binding site/antigen complex formation and dissociation, where these rates depend on the concentration of complex partners, affinity of interactions, and geometric parameters that affect the rate equally in both directions. Thus, "binding constant" (K on ) And "dissociation constant" (K) off ) Both can be determined by calculating the concentration and the actual association and dissociation rates. (see Malmqvist, nature 361:186-187 (1993)). K (K) off /K on Is capable of eliminating all affinity-independent parameters and is equal to the dissociation constant K d . (see Davies et al (1990) Annual Rev Biochem 59:59:439-473). When the equilibrium binding constant (K d ) An antibody or antigen-binding portion provided herein is considered to specifically bind CD122 at 10. Mu.M.ltoreq.M, preferably 10nM, more preferably 10nM, and most preferably 100pM to about 1 pM. K for determining antibodies d By using Surface Plasmon Resonance (SPR), typically using a biosensor system such asThe system.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein is monovalent or bivalent, and comprises a single or double strand. Functionally, the binding affinity of the antibody or antigen binding portion may be at 10 -5 M to 10 -12 M is in the range of M. For example, the binding affinity of the antibody or antigen binding portion is 10 -6 M to 10 -12 M、10 -7 M to 10 -12 M、10 -8 M to 10 - 12 M、10 -9 M to 10 -12 M、10 -5 M to 10 -11 M、10 -6 M to 10 -11 M、10 -7 M to 10 -11 M、10 -8 M to 10 -11 M、10 -9 M to 10 -11 M、10 -10 M to 10 -11 M、10 -5 M to 10 -10 M、10 -6 M to 10 -10 M、10 -7 M to 10 -10 M、10 -8 M to 10 -10 M、10 -9 M to 10 -10 M、10 -5 M to 10 -9 M、10 -6 M to 10 -9 M、10 -7 M to 10 -9 M、10 -8 M to 10 -9 M、10 -5 M to 10 -8 M、10 -6 M to 10 -8 M、10 -7 M to 10 -8 M、10 -5 M to 10 -7 M、10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M。
Provided herein is an anti-CD 122 antibody, or antigen-binding portion thereof, wherein the antibody or antigen-binding portion cross-competes for binding to CD122 with antibody MAB05 or MAB06, or an antibody comprising one or more amino acid sequences of antibody MAB05 or MAB06 (see tables 18 and 19).
The terms "cross-compete", "cross-block" and "cross-block" are used interchangeably herein to refer to the ability of an antibody or antigen-binding portion thereof to interfere directly or indirectly with binding through allosteric modulation of target CD122 (e.g., human CD 122) by an anti-CD 122 antibody of the present disclosure. The extent to which an antibody or portion thereof is able to interfere with the binding of another antibody to a target, and thus whether it can be considered cross-blocking or cross-competing, can be determined using a competitive binding assay. One example of a binding competition assay is Homogeneous Time Resolved Fluorescence (HTRF). A particularly suitable quantitative cross-competition assay uses FACS or Alphascreen-based methods to measure competition between a labelled (e.g. His-tagged, biotinylated or radiolabeled) antibody or part thereof and another antibody or part thereof according to their binding to a target. Typically, the cross-competing antibody or portion thereof is the following antibody: for example, will bind to a target in a cross-competition assay such that during the assay and in the presence of a second antibody or portion thereof, the recorded shift of an immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g. in a FACS-based competition assay) by potentially cross-blocking the maximum theoretical shift caused by an antibody or fragment thereof present in a given amount (e.g. shift caused by a cold (e.g. unlabeled) antibody or fragment thereof that needs to be cross-blocked). In some embodiments, the cross-competing antibody, or portion thereof, has a recorded shift between 10% and 100%, or between 50% and 100%.
Provided herein is an anti-CD 122 antibody, or antigen-binding portion thereof, wherein the antibody or antigen-binding portion cross-competes for binding to CD122 with an antibody comprising a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion cross-competes for binding to CD122 with an antibody or antigen-binding portion comprising a set of CDRs disclosed herein; and (a) specifically binds to (i) human CD122 and (ii) cynomolgus monkey and/or rhesus CD122; (b) Antagonizing human CD122 with an EC50 of less than 14nM when stimulated by human IL-15 + Proliferation of cells (such as primary NK cells); (c) binds rhesus CD122 with a KD of less than 10 nM; (d) Binding to functionally identical epitopes on cynomolgus monkey and/or rhesus monkey CD122 and human CD122; and/or (e) exhibits no or reduced BCAM binding as compared to an anti-CD 122 antibody comprising the variable domain sequence of antibody mikβ1. In some embodiments, the antibody or antigen binding portion of K d The value may be byAnalysis to determine. In some embodiments, the EC50 value of an antibody or antigen binding portion can be determined by flow cytometry staining of CD122 expressing cells (e.g., kacho cells, HEK cells, M07e cells, NK cells, T cells).
In some embodiments, the anti-CD 122 antibodies or antigen-binding portions provided herein have low immunogenicity. In some embodiments, the antibody or antigen binding portion exhibits reduced immunogenicity as compared to an anti-CD 122 antibody comprising HCDR1 of SYGVH (SEQ ID NO: 24), HCDR2 of VIWSGGSTDYNAAFIS (SEQ ID NO: 5), HCDR3 of AGDYNYDGFAY (SEQ ID NO: 27), LCDR1 of SGSSSVSFMY (SEQ ID NO: 30), LCDR2 of DTSNLAS (SEQ ID NO: 13) and LCDR3 of QQWSTYPLT (SEQ ID NO: 15). In some examples, the risk of immunogenicity of an antibody or antigen binding portion can be determined in silico by identifying the location of a T cell epitope in the antibody or portion (e.g., in the variable region of the antibody or portion).
For example, T cell epitopes in antibodies or antigen binding portions can be detected by using iTope TM To identify. iTope TM Peptides with promiscuous high affinity binding to human MHC class II that can be used to analyze VL and VH region sequences. Promiscuous high affinity MHC class II binding peptides are thought to be associated with the presence of T cell epitopes, which are a high risk indicator of clinical immunogenicity of pharmaceutical proteins. iTope TM The software predicts an advantageous interaction between the amino acid side chains of the peptide and specific binding pockets (in particular pocket positions; p1, p4, p6, p7 and p 9) within the open binding groove of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide, with weights not attributed to those most commonly found in any particular ethnic group. Twenty alleles contain an "open" p1 configuration and 14 alleles contain a "closed" configuration, with glycine at position 83 replaced with valine. The position of the critical binding residues is achieved by generating in silico a 9mer peptide that overlaps eight amino acids across the test protein sequence. The method is successfulPeptides that bind or do not bind to MHC class II molecules are distinguished with high accuracy.
T cell epitopes in antibodies or antigen binding portions can be obtained by using TCED TM (T cell epitope database) TM ) VL region and VH region sequences were analyzed to find matches to T cell epitopes previously identified by in vitro human T cell epitope mapping analysis of other protein sequences. TCED TM For searching a large (> 10,000 peptides) database of peptides derived from unrelated proteins and antibody sequences for any test sequence.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion may exhibit low immunogenicity because the antibody or portion has a low amount of one or more of the following peptides in its sequence: high affinity foreign peptides ("HAF" -high immunogenicity risk), low affinity foreign peptides ("LAF" -lower immunogenicity risk), and/or tced+ (previously in TCED) TM Epitopes identified in the database).
In some embodiments, an anti-CD 122 antibody or antigen-binding portion may have a high germ line epitope (GE) content in its sequence. In some examples, an anti-CD 122 antibody or antigen-binding portion has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or greater than 20) germline epitopes in its sequence (e.g., in the VL region and/or VH region sequences). Germline epitopes can be defined as human germline peptide sequences with high MHC class II binding affinity. Germline epitope 9mer peptides are unlikely to have immunogenic potential due to T cell tolerance, as has been demonstrated by previous studies using a variety of germline peptides. Importantly, such germline v-domain epitopes (further aided by similar sequences in the human antibody constant region) also compete for MHC class II occupancy on antigen presenting cell membranes, thereby reducing the risk of exogenous peptide presentation sufficient to reach the "threshold of activation" required for T cell stimulation. Thus, high GE content is a beneficial quality in the clinical development of antibody therapeutics and can provide low immunogenicity. In some examples, the anti-CD 122 antibody or antigen-binding portion comprises a human germline peptide sequence (e.g., germline epitope) with high MHC class II binding affinity in LCDR 2.
In certain embodiments, the anti-CD 122 antibody or antigen-binding portion may have a reduced number of HAF, LAF, and/or tced+ epitopes found in the framework of both the heavy and light chain variable regions as compared to an anti-CD 122 antibody comprising the variable domain sequence of antibody mikβ1. In some embodiments, the HAF, LAF, and/or tced+ epitope is not present in the VL region and/or VH region sequence of the anti-CD 122 antibody or antigen binding portion.
In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof, does not comprise one or more mikβ1 murine/humanized antibody amino acid sequences provided in table 20. In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof, does not comprise a polypeptide comprising SEQ ID NO:24, HCDR1 comprising SEQ ID NO:27 and/or HCDR3 comprising SEQ ID NO: LCDR1 of 30. Table 1 provides the amino acid sequences of the variable regions of MIKβ1 murine anti-CD 122 antibodies, wherein the highlighted CDRs are as defined herein ("Kabat" protocol). The term "MIKβ1-IgG1 (humanized)" refers to an anti-CD 122 antibody comprising the variable heavy chain region sequence labeled CD122-VH1 and the variable light chain region sequence labeled CD122-VL1 in Table 2, as well as human IgG1 constant regions.
The antibodies disclosed herein are anti-CD 122 antagonist antibodies. As used herein, an "antagonist" or "anti-CD 122 antagonist antibody" (interchangeably referred to as "anti-CD 122 antibody") refers to an antibody that is capable of binding CD122 and inhibiting CD122 biological activity and/or downstream pathways mediated by CD122 signaling. anti-CD 122 antagonist antibodies encompass antibodies that can block, antagonize, suppress, or reduce (including significantly reduce) CD122 biological activity (including downstream pathways mediated by CD122 signaling, such as receptor binding and/or eliciting a cellular response to CD 122). For the purposes of this disclosure, it is to be expressly understood that the term "anti-CD 122 antagonist antibody" encompasses all terms, names and functional states and features in which CD122 itself and CD122 biological activity (including but not limited to its ability to inhibit activation of anti-tumor cell activity of T cells) or the result of activity or biological activity is substantially ineffective, reduced or neutralized to any meaningful extent.
In some embodiments, the antibody molecule, or antigen-binding portion thereof, specifically binds CD122, but does not bind (or does not specifically bind) membrane protein BCAM. In some embodiments, the BCAM is a human protein. In some embodiments, the BCAM is a rhesus protein. In some embodiments, the human BCAM protein comprises or consists of: amino acid sequence SEQ ID NO:21 or with SEQ ID NO:21 has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In one embodiment, the antibody molecule or antigen binding portion thereof does not bind BCAM. In some embodiments, the binding of the antibody molecule or antigen binding portion thereof to BCAM is reduced compared to the binding of the antibody mikβ1 or IgG1-mikβ1 (humanized) to the membrane receptor. In some cases, the binding of the antibody or antigen-binding portion thereof to the BCAM can be determined by ELISA or flow cytometry analysis.
Also provided herein is an anti-CD 122 antibody, or antigen-binding portion thereof, comprising one or more amino acid sequences of antibody MAB06 or MAB 05. Tables 18 and 19 provide combinations of VH, VL and CDR sequences that form these antibodies. In some embodiments, the VH region sequence and/or the VL region sequence comprises a signal sequence (also referred to as a signal peptide) at the amino terminus.
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO:7 and the VL region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO:15, or (b) the VH region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO:7 and the VL region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:11, LCDR1, containing SEq ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
In some embodiments, disclosed herein are anti-CD 122 antibodies, or antigen-binding portions thereof, wherein the antibodies comprise a polypeptide comprising HCDR1, HVH regions of CDR2 and HCDR3 and VL regions comprising LCDR1, LCDR2 and LCDR3, wherein (a) said HCDR1 comprises the amino acid sequence G-F-T-F-S-Y-X 1 -M-S, wherein X 1 Is L or any other amino acid (SEQ ID NO: 39); (b) The HCDR2 comprises amino acid sequence X 1 -A-X 2 -I-S-G-G-G-X 3 -X 4 -X 5 -Y-Y-X 6 -D-S-V-K-G, wherein X 1 Is V or a conservative substitution of V, X 2 Is T or N, X 3 Is A or S, X 4 Is E or N, X 5 Is T or K, and X 6 Is P or V (SEQ ID NO: 40); (c) The HCDR3 comprises amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -D-Y, wherein X 1 Is Q or any other amino acid (e.g., T, L, M or N), X 2 Is L or any other amino acid (e.g., G, K, M, Q, S or V), X 3 Is Y or a conservative substitution of Y (e.g., H), X 4 Is Y or any other amino acid (e.g., A, D, F, G, M, E, I, K, S or W) and X 5 Is F or any other amino acid (e.g., A, D, E, I, K, M, S or W) (SEQ ID NO: 160); (d) The LCDR1 comprises the amino acid sequence R-A-S-q-S-I-X 1 -X 2 -X 3 -X 4 -X 5 Wherein X is 1 Is S or a conservative substitution of S, X 2 Is S or a conservative substitution of S, X 3 Is Y or a conservative substitution of Y, X 4 Is L or a conservative substitution of L, and X 5 Is N or T or a conservative substitution of N or T (SEQ ID NO: 161); (e) The LCDR2 comprises amino acid sequence X 1 -A-X 2 -S-L-X 3 -X 4 Wherein X is 1 Is A or T or a conservative substitution of A or T, X 2 Is S or a conservative substitution of S, X 3 Is Q or any other amino acid, and X 4 Is S or any other amino acid (SEQ ID NO: 162); and (f) said LCDR3 comprises the amino acid sequence Q-Q-X 1 -Y-S-X 2 -P-X 3 -T, wherein X 1 Is S or any other amino acid, X 2 Is T or any other amino acid, and X 3 Is W or any other amino acid (SEQ ID NO: 163).
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEq ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consist thereof.
Provided herein is an anti-CD 122 antibody, or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein the VH region amino acid sequence comprises an amino acid sequence of SEO ID NO:1 or with the amino acid sequence SEQ ID NO:1 has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical.
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein the VL region amino acid sequence comprises (a) SEQ ID NO:17 or with the amino acid sequence SEQ ID NO:9 has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical; or (b) SEQ ID NO:17 or with the amino acid sequence SEQ ID NO:9 has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical.
Also provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein (a) the VH region amino acid sequence comprises SEQ ID NO:1 or with the amino acid sequence SEQ ID NO:1 having an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical; and the VL region amino acid sequence comprises SEQ ID NO:17 or with the amino acid sequence SEQ ID NO:9 has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or with the amino acid sequence SEQ ID NO:1 having an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical; and the VL region amino acid sequence comprises SEQ ID NO:9 or with the amino acid sequence SEQ ID NO:17 has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical.
Provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein (a) the VH region amino acid sequence comprises SEQ ID NO:1, a step of; and the VL region amino acid sequence comprises SEQ ID NO:17, wherein there are 1, 2 or 3 conservative amino acid substitutions in the VH region sequence, the VL region sequence, or both the VH and VL region sequences; or (b) the VH region amino acid sequence comprises SEQ ID NO:1, a step of; and the VL region amino acid sequence comprises SEQ ID NO:9, wherein there are 1, 2 or 3 conservative amino acid substitutions in the VH region sequence, the VL region sequence, or both the VH and VL region sequences. In some embodiments, conservative amino acid substitutions are made only in the FR sequence of the antibody or antigen binding portion, and not in the CDR sequence.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein is monoclonal. The term "monoclonal antibody" (Mab) refers to an antibody or antigen-binding portion thereof that is derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), rather than from the method by which it was produced. Preferably, the monoclonal antibodies are present in a homogeneous or substantially homogeneous population.
In some embodiments, the antibodies or antigen binding portions provided herein can be isolated.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein is chimeric. The term "chimeric" refers to an antibody molecule or antigen binding portion thereof in which the variable domain sequence is derived from one species and at least one constant region sequence is derived from another species. For example, one or all of the variable domains of the light chain and/or one or all of the variable domains of the heavy chain of a mouse antibody (e.g., a mouse monoclonal antibody) may each be linked to a human constant region, such as, but not limited to, an IgG1 or IgG4 human constant region. U.S.4,816,567; U.S.4,975,369; examples of chimeric antibodies and suitable techniques for producing chimeric antibodies are provided in U.S. Pat. No. 4,816,397, each of which is incorporated herein by reference in its entirety. In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein comprises: (a) a polypeptide comprising SEQ ID NO:1, a VH region amino acid sequence; comprising SEQ ID NO:17 and a human constant region; or (b) a polypeptide comprising SEQ ID NO:1, comprising the VH region amino acid sequence of SEQ ID NO:9 and a human constant region.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein is humanized. The term "humanized" refers to antibodies or antigen-binding portions thereof that have been engineered to comprise one or more human framework regions in the variable domain and non-human (e.g., mouse, rat, or hamster) CDRs of the heavy and/or light chain. In some embodiments, the humanized antibody comprises sequences that are fully human except for the CDRs. The anti-CD 122 antibody molecule or antigen binding portion thereof may comprise one or more human variable region framework scaffolds into which CDRs have been inserted. In some embodiments, the VH region, VL region, or both VH and VL regions of an anti-CD 122 antibody or antigen-binding portion provided herein comprise one or more human framework region amino acid sequences. In some embodiments, the humanized antibody comprises sequences that are fully human except for the CDRs, which are CDRs of antibody MAB06 or MAB 05. Examples of humanized antibodies and suitable generation techniques therefor are provided in Hwang et al, methods 36:35 2005, 2005; queen et al, proc. Natl. Acad. Sci. USA,86:10029-10033, 1989; jones et al, nature,321:522-25, 1986; riechmann et al, nature,332:323-27, 1988; verhoeyen et al Science,239:1534-36, 1988; orlandi et al, proc.Natl. Acad.Sci.USA,86:3833-37, 1989; U.S.5,225,539; U.S.5,530,101; U.S.5,585,089; U.S.5,693,761; U.S.5,693,762; U.S.6,180,370; and WO 90/07861, each of which is incorporated herein by reference in its entirety. When selecting FR flanking CDRs, for example when humanizing or optimizing an antibody, it is preferred that the FR is from an antibody containing CDR sequences of the same canonical class.
In some embodiments, an anti-CD 122 antibody or antigen-binding fragment provided herein does not necessarily have the maximum number of human germline substitutions at the corresponding murine CDR or other (such as framework) amino acid positions. As described in the experimental section below, the "maximizing the humanized" antibody molecules are not necessarily "maximally optimized" in terms of anti-CD 122 binding properties and/or other desired characteristics.
The present disclosure includes modifications to the amino acid sequence of an antibody molecule or antigen binding portion thereof as defined herein. For example, the present disclosure includes antibody molecules and corresponding antigen binding portions thereof that include functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with enhanced or reduced activity and/or affinity. For example, the amino acid sequence may be mutated to obtain an antibody having a desired binding affinity for CD 122. Insertions, including amino-terminal and/or carboxy-terminal fusions, ranging in length from one residue to polypeptides containing one hundred or more residues, and intra-sequence insertions of single or multiple amino acid residues are contemplated. Examples of terminal insertions include antibody molecules having an N-terminal methionyl residue or antibody molecules fused to an epitope tag. Other insertional variants of antibody molecules include fusions of enzymes or polypeptides with the N-or C-terminus of an antibody that increase the half-life of the antibody in the blood circulation.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein can include glycosylated and non-glycosylated polypeptides, as well as polypeptides having other post-translational modifications, such as glycosylation, acetylation, and phosphorylation with different sugars. Antibodies or antigen binding portions may be mutated to alter such post-translational modifications, for example by adding, removing, or replacing one or more amino acid residues to form or remove glycosylation sites.
In some embodiments, an anti-CD 122 antibody or antigen-binding portion provided herein can be modified, for example, by amino acid substitution to remove potential proteolytic sites in the antibody or portion.
Also provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region, a VL region, and all human framework region sequences, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
Also provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region, a VL region, and one or more human framework region sequences, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof, may comprise an IGHV3-23 human germline scaffold into which the corresponding HCDR sequence has been inserted. The anti-CD 122 antibody, or antigen-binding portion thereof, may comprise a VH region comprising IGHV3-23 human germline scaffold amino acid sequences into which a set of corresponding HCDR1, HCDR2 and HCDR3 amino acid sequences have been inserted.
In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof, may comprise an IGKV1-33 human germline scaffold into which the corresponding LCDR sequence has been inserted. The anti-CD 122 antibody, or antigen-binding portion thereof, may comprise a VL region comprising an IGKV1-33 human germline scaffold amino acid sequence into which a set of corresponding LCDR1, LCDR2, and LCDR3 amino acid sequences have been inserted.
In some embodiments, the anti-CD 122 antibody or antigen-binding portion thereof may comprise an IGHV3-23 human germline scaffold into which the corresponding HCDR sequence has been inserted and an IGKV1-33 human germline scaffold into which the corresponding LCDR sequence has been inserted. The anti-CD 122 antibody, or antigen-binding portion thereof, may comprise a VH region comprising an IGHV3-23 human germline scaffold amino acid sequence into which a set of corresponding HCDR1, HCDR2 and HCDR3 amino acid sequences have been inserted, and a VL region comprising an IGKV1-33 human germline scaffold amino acid sequence into which a set of corresponding LCDR1, LCDR2 and LCDR3 amino acid sequences have been inserted. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences may be HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of any one of table 18 or 19 (all six CDR sequences are from the same clone).
In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof, comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG, igE, igM, igD, igA or IgY. In some embodiments, the immunoglobulin constant region is IgG1, igG2, igG3, igG4, igA1, or IgA2. In some embodiments, the immunoglobulin constant region is immunologically inert. In some embodiments, the immunoglobulin constant region comprises one or more mutations that reduce or prevent fcγr binding, antibody dependent cell-mediated cytotoxic activity, and/or complement dependent cytotoxic activity. In some embodiments, the immunoglobulin constant region is a wild-type human IgG1 constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgG1 constant region comprising amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A and P331S, or a human IgG4 constant region comprising amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat. In some embodiments, the positions of amino acid residues in the constant region of an immunoglobulin molecule are numbered t according to the EU index as in Kabat (Ward et al, 1995 Therap.Immunol.2:77-94).
In some embodiments, an anti-CD 122 antibody, or antigen-binding portion thereof, may comprise an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.
In some embodiments, an anti-CD 122 antibody may comprise an immunoglobulin constant region comprising any of the amino acid sequences in table 15. The Fc region sequences in table 15 begin with the CH1 domain. In some embodiments, an anti-CD 122 antibody may comprise an immunoglobulin constant region comprising an amino acid sequence of a human IgG4, human IgG4 (S228P), human IgG2, human IgG1, fc region in which human IgG1 effectors are ineffective. For example, the human IgG4 (S228P) Fc region comprises the following substitutions compared to the wild-type human IgG4 Fc region: S228P. For example, the human IgG1 effector-null Fc region comprises the following substitutions compared to the wild-type human IgG1 Fc region: L234A, L a and G237A. In some embodiments, the immunoglobulin constant region may comprise an RDELT (SEQ ID NO: 39) motif or a REEM (SEQ ID NO: 40) motif (underlined in Table 15). REEM (SEQ ID NO: 40) allotype is found in smaller populations than RDELT (SEQ ID NO: 39) allotype. In some embodiments, an anti-CD 122 antibody may comprise an immunoglobulin constant region comprising the amino acid sequence of SEQ ID NO: 32-38. In some embodiments, an anti-CD 122 antibody may comprise six CDR amino acid sequences of any clone of table 18 or 19 and any Fc region amino acid sequence of table 15. In some embodiments, an anti-CD 122 antibody may comprise an immunoglobulin heavy chain constant region comprising any of the Fc region amino acid sequences of table 15 and an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.
In some embodiments, provided herein is an anti-CD 122 antibody, or antigen-binding portion thereof, wherein the antibody comprises a VH region, a VL region, and a heavy chain constant region, wherein (a) the VH region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; and the heavy chain constant region comprises SEQ ID NO: 32-38; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; and the heavy chain constant region comprises SEQ ID NO: 32-38.
In some embodiments, provided herein is an anti-CD 122 antibody, or antigen-binding portion thereof, wherein the antibody comprises a VH region, a VL region, and a heavy chain constant region, wherein (a) the VH region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; and the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, and a wild-type human IgG2 constant region; wild type human IgG1 constant region or a human IgG1 constant region comprising amino acid substitutions L234A, L235A and G237A; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; and the heavy chain constant region comprises SEQ ID NO: 32-38; and the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, and a wild-type human IgG2 constant region; wild-type human IgG1 constant region or human IgG1 constant region comprising amino acid substitutions L234A, L235A and G237A.
In some embodiments, provided herein is an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists of the same; the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; and the heavy chain constant region comprises SEQ ID NO: 32-38; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists of the same; the VL region amino acid sequence comprises SEQ ID NO:9 or consists thereof; and the heavy chain constant region comprises SEQ ID NO: 32-38.
In some embodiments, the anti-CD 122 antibody may be immune effector-null. In some embodiments, the anti-CD 122 antibody or antigen-binding portion thereof does not induce immune effector function, and optionally inhibits immune effector function. In some embodiments, an anti-CD 122 antibody may lack measurable binding to human fcyri, fcyriia, fcyriiia, and fcyriiib receptors, but maintain binding to human fcyriib receptors, and optionally to human FcRn receptor. Fcyri, fcyriia, fcyriiia, and fcyriiib are examples of activating receptors. Fcyriib is an example of an inhibitory receptor. FcRn is an example of a recycling acceptor. In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof Binding affinity for human Fc receptors can be achieved byAnalysis to measure. In some embodiments, homogeneous Time Resolved Fluorescence (HTRF) may be used to study the binding of anti-CD 122 antibodies to human Fc receptors. In one example of HTRF, human IgG1 (wild type) is labeled, the whole set of fcγ receptors is also labeled, and then antibodies with engineered Fc fragments are used to titrate competition. In some embodiments, CD122 positive cells may be mixed with human leukocytes and anti-CD 122 antibodies, and cell killing by CDC, ADCC, and/or ADCP may be measured. In some embodiments, an anti-CD 122 antibody (see table 15) comprising the amino acid sequence of the Fc region of human IgG1 is effector-null. In some embodiments, an anti-CD 122 antibody comprising the amino acid sequence of the Fc region of human IgG1 (see table 15) is not effector-null.
Also provided herein is an immunoconjugate comprising an anti-CD 122 antibody or antigen-binding portion thereof linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, radioisotope, chemotherapeutic agent, immunomodulatory agent, cytostatic enzyme, cytolytic enzyme, therapeutic nucleic acid, anti-angiogenic agent, anti-proliferative agent, or pro-apoptotic agent.
Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, anti-angiogenic agents, antiproliferatives, pro-apoptotic agents, and cytostatic and cytolytic enzymes (e.g., rnases). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, anti-angiogenic agents, antiproliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive and thus one or more of the above terms may be used to describe a therapeutic agent.
Examples of suitable therapeutic agents for immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansine, CC-1065 and sesqui-carcinomycin, spinosyns and other enediynes, and reoxygenate (auristatin). Other examples include antifolates, vinca alkaloids and anthracyclines. Phytotoxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers may also be used in the immunoconjugate. In addition, conjugates can be prepared using a secondary carrier as a cytotoxic agent (such as a liposome or polymer). Suitable cytotoxins include agents that inhibit or prevent cellular function and/or cause cellular destruction. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and disrupt DNA, and agents that disrupt protein synthesis or function of essential cellular proteins such as protein kinases, phosphatases, topoisomes, enzymes, and cyclin.
Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, doxorubicin, zorubicin, mitoxantrone, epirubicin, cartrubicin, noramycin, minoxidil, pirarubicin (pitarubicin), pentosrubicin, cytarabine, gemcitabine, trifluoracetamide, ancitabine, enocitabine, azacytidine, doxifluridine, penstatin, bromouridine (broxuridine), capecitabine, cladribine (cladbine), decitabine, fluorouridine (floxuridine), fludarabine, oryzanol, puromycin, tegafur, thiazolecarboxamide (tizofuhn), doxifuhn methacycline (adhamycin), cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, paclitaxel analogs, platinum species such as cisplatin and carboplatin, mitomycin, thiotepa, taxane, vincristine, daunorubicin, epirubicin, actinomycin, aflatoxin (authamycin), azoserine, bleomycin, tamoxifen, idarubicin, dolastatin/auristatin, hamitelin (hemiasterlin), epothilone, and maytansinoid.
Suitable immunomodulators include anti-hormonal agents that block the action of hormones on tumors, and immunosuppressants that inhibit cytokine production, down-regulate autoantigen expression, or mask MHC antigens.
Pharmaceutical composition
The anti-CD 122 antibodies and antigen-binding portions (also referred to herein as "active compounds") provided herein may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an anti-CD 122 antibody or antigen-binding portion (or an immunoconjugate comprising said antibody or portion), and a pharmaceutically acceptable carrier, diluent or excipient. Such materials should be non-toxic and should not interfere with the efficacy of the anti-CD 122 antibody or antigen-binding fragment thereof. The precise nature of the carrier or other material will depend on the route of administration, which may be injection, bolus injection, infusion or any other suitable route, as discussed below.
As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not normally produce allergies or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions for animals, more particularly for humans, approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia are considered "pharmaceutically acceptable". As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable vectors are described in the latest version of Remington's Pharmaceutical Sciences, a standard reference in the art, incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds may also be incorporated into the compositions. The pharmaceutically acceptable carrier, diluent or excipient may be a compound or combination of compounds that does not cause side effects and allows, for example, to facilitate administration of the anti-CD 122 antibody or antigen-binding portion thereof, increase its lifetime and/or efficacy in vivo, or increase its solubility in solution.
Provided herein is a pharmaceutical composition comprising (i) an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; and (ii) a pharmaceutically acceptable carrier, diluent or excipient; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
Provided herein is a pharmaceutical composition comprising (i) an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consists thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.
The pharmaceutical compositions disclosed herein may be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhaled), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may contain the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor (BASF, parippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid for easy injectability. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients as required and then sterilizing by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains an alkaline dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions typically comprise an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions may also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is applied orally and then rinsed, expectorated or swallowed. Pharmaceutically compatible binders and/or auxiliary materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid,Or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For inhaled administration, the compound may be delivered as an aerosol spray from a pressure vessel or dispenser containing a suitable propellant, such as a gas, e.g., carbon dioxide, or a nebulizer.
Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents for transmucosal administration, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, salves, gels, or creams, as generally known in the art.
The medicament may also be formulated in the form of suppositories (e.g. with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In some embodiments, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposomal suspensions may also be used as pharmaceutically acceptable carriers.
For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect with the desired pharmaceutical carrier. The specification of the dosage unit form of the invention is determined by, and directly depends on, the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the technology of compounding such active compounds for the treatment of individuals.
In some embodiments, the anti-CD 122 antibody, or antigen-binding portion thereof, may be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.
The pharmaceutical compositions provided herein may be contained in a container, package, or dispenser together with instructions for administration.
Nucleic acid molecules, vectors, host cells and methods for producing antibodies
Provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding the amino acid sequences of an anti-CD 122 antibody or anti-CD 122 antigen-binding portion described herein (or the amino acid sequences of (i) a VH region, (ii) a VL region, or (iii) both a VH region and a VL region of an antibody or antigen-binding portion). Also provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding (i) a heavy chain, (ii) a light chain, or (iii) both a heavy chain and a light chain of an anti-CD 122 antibody or anti-CD 122 antigen binding portion described herein. In some embodiments, the nucleic acid molecule encoding a VH region, VL region, heavy chain, or light chain comprises a signal sequence. In some embodiments, the nucleic acid molecule encoding a VH region, VL region, heavy chain, or light chain does not comprise a signal sequence.
In some embodiments, the nucleic acid molecule encodes the amino acid sequences of the VH and VL regions of an anti-CD 122 antibody or antigen-binding portion thereof, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15. In some embodiments, the nucleic acid molecule further encodes a human framework region amino acid sequence.
In some embodiments, the nucleic acid molecule encodes the amino acid sequences of the VH and VL regions of an anti-CD 122 antibody or antigen-binding portion thereof, wherein (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consist thereof.
Also provided herein is an expression vector comprising a nucleic acid molecule as described herein. In certain vectors, the nucleic acid molecule is operably linked to one or more control sequences suitable for allowing expression of the nucleic acid segment in a host cell. In some cases, the expression vector comprises a sequence that mediates replication and comprises one or more selectable markers. As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors; a naked DNA or RNA expression vector; plasmid, cosmid or phage vectors; a DNA or RNA expression vector associated with a cationic condensing agent; a DNA or RNA expression vector encapsulated in a liposome; and certain eukaryotic cells, such as producer cells.
Provided herein is a recombinant host cell comprising an expression vector or nucleic acid molecule disclosed herein. "host cells" include single cells, cell lines, or cell cultures that may or may not be recipients of the vector for incorporation of the polynucleotide insert. Host cells include progeny of a single host cell. Due to natural, accidental, or deliberate mutation, the offspring may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. The expression vector may be transfected into the host cell by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. In some embodiments, the recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both the VH region and the VL region of the anti-CD 122 antibody or antigen-binding portion thereof. In some embodiments, the recombinant host cell comprises (i) a first vector or first nucleic acid molecule encoding a VH region of an anti-CD 122 antibody or antigen-binding portion thereof, and (ii) a second vector or second nucleic acid molecule encoding a VL region of an anti-CD 122 antibody or antigen-binding portion thereof.
The antibody molecules of the invention, or antigen binding portions thereof, may be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, computational techniques, or combinations of such techniques, or other techniques readily known in the art.
Also provided herein is a method for producing an anti-CD 122 antibody, or antigen-binding portion thereof, comprising: culturing a recombinant host cell comprising an expression vector described herein under conditions that express the nucleic acid segment, thereby producing the anti-CD 122 antibody or antigen-binding portion. The antibody or antigen binding portion may then be isolated from the host cell or culture. anti-CD 122 antibodies and antigen-binding portions thereof may be produced by any of a variety of methods known to those of skill in the art. In certain embodiments, anti-CD 122 antibodies and antigen-binding portions thereof may be recombinantly produced. For example, a polypeptide encoding SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO:17 or SEQ ID NO:18 or a portion thereof into a bacterial cell (e.g., escherichia coli, bacillus subtilis) or eukaryotic cell (e.g., saccharomyces cerevisiae, for example, or mammalian cells such as CHO cell lines, various Cos cell lines, heLa cells, HEK293 cells, various myeloma cell lines, or transformed B cells or hybridomas), or into an in vitro translation system, and the translated polypeptides may be isolated. In some embodiments, the antibody light chain protein and heavy chain protein are produced in a cell, wherein the signal sequence is removed upon production of the mature anti-CD 122 antibody or antigen binding portion thereof.
One of skill in the art will be able to determine whether an antibody or antigen binding portion comprising a given polypeptide sequence binds to CD122 protein without undue experimentation using standard methods, such as western blotting, ELISA, and the like.
Provided herein is a method of producing an antibody that specifically binds to human CD122, and optionally also to cynomolgus monkey and/or rhesus monkey CD122, or an antigen binding portion thereof, comprising the steps of:
(1) Grafting anti-CD 122 CDRs from a non-human source into a human v-domain framework to produce a humanized anti-CD 122 antibody molecule or antigen-binding portion thereof;
(2) Generating a library of clones of humanized anti-CD 122 antibody molecules or antigen binding portions thereof, comprising one or more mutations in the CDRs;
(3) Screening a library that binds to human CD122 and optionally also to cynomolgus monkey and/or rhesus monkey CD 122;
(4) Selecting from the screening step (3) a clone having binding specificity for human CD122 and optionally for cynomolgus monkey and/or rhesus monkey CD122 but reduced binding or lack of binding to human BCAM, human CILP2 or human neudins; and
(5) Generating an antibody molecule that specifically binds to human CD122 and optionally also to cynomolgus monkey and/or rhesus monkey CD122 or antigen binding portions thereof from the clone selected from step (4).
The method may comprise the further step of: based on the clone selected in step (4), e.g. based on further exploratory mutagenesis of a specific position in the CDRs of the clone selected in step (4), additional clones are generated to enhance humanization and/or minimize the content of human T cell epitopes and/or to improve the manufacturing properties of the antibody molecule or antigen binding portion thereof produced in step (5).
Use of antibodies
Provided herein are methods and uses of the anti-CD 122 antibodies, anti-CD 122 antigen-binding portions, immunoconjugates and pharmaceutical compositions described herein for providing therapeutic benefit to a subject suffering from an immune-mediated disease or disorder.
Provided herein is a method for inhibiting an immune response in a subject comprising administering to the subject a therapeutically effective amount of an antibody, antigen binding portion, immunoconjugate or pharmaceutical composition disclosed herein. Provided herein is a method for inhibiting an immune response in a subject (e.g., an immune response mediated by CD122 positive cells), the method comprising administering to the subject a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15. Provided herein is a method for inhibiting an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consist thereof. In some embodiments, the immune response is mediated by CD 122.
Provided herein is a method for inhibiting IL-15-induced T cell migration from skin (e.g., human skin) comprising contacting the skin with a therapeutically effective amount of an antibody, antigen-binding portion, immunoconjugate or pharmaceutical composition disclosed herein. Provided herein is a method for inhibiting IL-15-induced T cell migration from skin (e.g., human skin), the method comprising contacting the skin with a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15. Provided herein is a method for inhibiting IL-15-induced T cell migration from skin (e.g., human skin), the method comprising contacting the skin with a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consist thereof. In some embodiments, the T cell is a cd8+ T cell. In some embodiments, the T cell is a cd4+ T cell. In some embodiments, the skin is the skin of a subject suffering from a disease or disorder associated with CD122 expression on cells that overexpress CD122 or that normally do not express CD 122.
The anti-CD 122 antibodies, or antigen-binding portions thereof, described herein can be used in methods of treating the human or animal body, including prophylactic or anti-predictive treatment (e.g., treatment prior to the onset of a subject condition to reduce the risk of developing a subject condition, delay the onset thereof, or reduce the severity thereof after the onset). The method of treatment may comprise administering an anti-CD 122 antibody or antigen-binding portion to a subject in need thereof. Provided herein is a method for treating or preventing a disease in a subject comprising administering to the subject a therapeutically effective amount of an antibody, antigen-binding portion, immunoconjugate, or pharmaceutical composition disclosed herein.
Provided herein is a method for treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15. Provided herein is a method for treating or preventing a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consist thereof.
Provided herein is a method for ameliorating, treating, or reducing the severity of a symptom of a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or (b) the VH region amino acid sequence comprises a sequence comprising SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15. Provided herein is a method for ameliorating, treating, or reducing the severity of a symptom of a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a VH region and a VL region, wherein: (a) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:17 or consist thereof; or (b) the VH region amino acid sequence comprises SEQ ID NO:1 or consists thereof, and the VL region amino acid sequence comprises SEQ ID NO:9 or consist thereof.
In some embodiments, the disease or disorder is associated with over-expression of CD122 or CD122 expression on cells that normally do not express CD 122. In some embodiments, the disease or disorder is mediated by CD 122.
In some embodiments, the disease is an inflammatory disease or an autoimmune disease. In some embodiments, the disease is vitiligo, celiac disease, type 1 diabetes, multiple sclerosis, graft versus host disease, systemic lupus erythematosus, psoriasis, atopic dermatitis, alopecia areata, ulcerative colitis, or rheumatoid arthritis.
In some embodiments, the VH region, VL region, or both VH and VL regions of an anti-CD 122 antibody or antigen-binding portion used in the methods provided herein comprise one or more human framework region amino acid sequences.
As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent, e.g., an anti-CD 122 antibody or antigen-binding portion thereof, sufficient to reduce or ameliorate the severity and/or duration of a disease or one or more symptoms thereof, e.g., vitiligo, celiac disease, type 1 diabetes, multiple sclerosis, graft versus host disease, systemic lupus erythematosus, psoriasis, atopic dermatitis, alopecia areata, ulcerative colitis, or rheumatoid arthritis; preventing disease progression; causing regression of the disease; preventing recurrence, development, onset or progression of one or more symptoms associated with the disease; detecting a disease; or enhance or improve the prophylactic or therapeutic effect of another related therapy for a CD122 mediated disease (e.g., a prophylactic or therapeutic agent).
The actual amount administered, as well as the rate and schedule of administration, will depend on the nature and severity of the disease to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the timing of administration, and other factors known to the practitioner. The prescription of treatment (e.g., decision on dosage, etc.) is the responsibility of the general practitioner and other doctors, and may depend on the severity and/or progression of the symptoms of the disease to be treated. Suitable dosages of antibody molecules are well known in the art (Ledermnn J.A. et al, 1991,Int.J.Cancer 47:659-664; bagshawe K.D. et al, 1991,Antibody,Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as dosages appropriate for the type of drug being administered may be used. A therapeutically effective amount or suitable dose of an antibody molecule can be determined by comparing its in vitro activity to its in vivo activity in an animal model. Methods of extrapolating effective dosages in mice and other test animals to humans are known. The precise dosage will depend on a number of factors including whether the antibody is for prophylaxis or therapy, the size and location of the area to be treated, the precise nature of the antibody (e.g., intact antibody, fragment), and the nature of any detectable label or other molecule attached to the antibody.
Typical antibody doses are in the range of 100 μg to 1g for systemic application and 1 μg to 1mg for intradermal injection. An initial higher loading dose may be administered followed by one or more lower doses. In some embodiments, the antibody is an intact antibody, e.g., an IgG1 or IgG4 isotype. This is a dose for a single treatment of adult subjects, which can be scaled up for children and infants, and also for other antibody formats, in proportion to molecular weight. The treatment may be repeated at daily, twice weekly, weekly or monthly intervals, at the discretion of the physician. The treatment regimen of a subject may depend on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration, and the nature of the condition being treated.
The treatment may be periodic and the period of time between administrations may be about two weeks or more, such as about three weeks or more, about four weeks or more, about one month or more, about five weeks or more, or about six weeks or more. For example, the treatment may be every 2-4 weeks or every 4-8 weeks. The treatment may be administered before and/or after surgery and/or may be administered or applied directly to the anatomical site of the surgical treatment or invasive surgery. Suitable formulations and routes of administration are as described above.
In some embodiments, the anti-CD 122 antibody molecules and antigen-binding portions described herein may be administered as subcutaneous injections. Subcutaneous injections may be made using an automatic injector, for example for long term prophylaxis/treatment.
In some embodiments, the therapeutic effect of an anti-CD 122 antibody or antigen-binding portion thereof may last for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of an anti-CD 122 antibody or antigen-binding portion thereof may last for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more in a subject.
In some embodiments, the subject may be treated with an anti-CD 122 antibody or anti-CD 122 antigen binding portion, an immunoconjugate or pharmaceutical composition described herein, or an additional therapeutic agent or therapy for treating a CD 122-mediated disease or disorder or a symptom or complication of a CD 122-mediated disease or disorder. The anti-CD 122 antibody or anti-CD 122 antigen binding portion and the additional therapeutic agent or therapy may be administered simultaneously or sequentially.
In some embodiments, the subject is a human, non-human primate, pig, horse, cow, dog, cat, guinea pig, mouse or rat. In some embodiments, the subject is an adult. In some embodiments, the subject is a pediatric human.
Also provided herein is an anti-CD 122 antibody or anti-CD 122 antigen binding portion, immunoconjugate or pharmaceutical composition described herein for use in treating a disease or disorder.
Provided herein is an anti-CD 122 antibody or anti-CD 122 antigen binding portion, immunoconjugate or pharmaceutical composition described herein for use as a medicament.
Definition of the definition
Unless otherwise indicated, terms used herein have the definitions commonly used in the art. Some terms are defined as follows, and additional definitions may be found in the remainder of the detailed description.
The terms "a" or "an" refer to one or more of the entity, i.e., a plurality of indicators. Thus, the terms "a", "an", "one or more", and "at least one" are used interchangeably herein. Furthermore, the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element.
Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of a device or method used to determine the value, or the variation present in a sample being measured. Unless otherwise indicated or apparent from the context, the term "about" means within 10% of the reported value (unless the value exceeds 100% of the possible value or is below 0%). Unless otherwise indicated, when used in connection with a range or series of values, the term "about" applies to the end of the range or each value recited in the series. As used in this application, the terms "about" and "about" are used as equivalents.
As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are unchanged throughout the residue (e.g., nucleotide or amino acid) alignment window. The "identity score" of an aligned segment of a test sequence and a reference sequence is the number of identical residues common to both aligned sequences divided by the total number of residues in the reference sequence fragment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. "percent identity" is the identity score multiplied by 100. The percentage identity can be calculated using default parameters using the alignment program Clustal Omega obtained at ebi.ac. uk/Tools/msa/clustalo. See Sievers et al, "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (2011, 10, 11 days) Molecular systems biology 7:539. to calculate sequence identity, extensions, such as tags, are not included.
As used herein, the term "HCDR" refers to the heavy chain complementarity determining region. As used herein, the term "LCDR" refers to the light chain complementarity determining region.
As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid that does not significantly deleteriously alter functional activity. A preferred example of a "conservative substitution" is the substitution of one amino acid with another amino acid having a value of.gtoreq.0 in the BLOSUM 62 substitution matrix below (see Henikoff and Henikoff,1992,PNAS 89:10915-10919):
"antibody-drug conjugate" and "immunoconjugate" refer to an antibody molecule or antigen-binding portion thereof, including antibody derivatives, that binds to CD122 and is conjugated to a cytotoxic, cytostatic, and/or therapeutic agent.
The term "isolated molecule" (wherein the molecule is, for example, a polypeptide, polynucleotide, or antibody) is a molecule that, due to its source or derived source, (1) is not associated with a naturally associated component that accompanies it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) is not found in nature. Thus, molecules that are chemically synthesized or expressed in a cellular system that differs from the cells from which they are naturally derived will be "isolated" from their naturally associated components. The molecules may also be substantially free of naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of means well known in the art. For example, the purity of a polypeptide sample may be determined using polyacrylamide gel electrophoresis, and the gel stained using techniques well known in the art to visualize the polypeptide. For some purposes, higher resolution may be provided by using HPLC or other means for purification known in the art.
The term "epitope" refers to a portion of a molecule that is capable of being recognized and bound by an antibody molecule or antigen binding portion thereof at one or more antigen binding regions of an antibody molecule. An epitope may consist of a defined region of a primary, secondary or tertiary protein structure and include a secondary structural unit or combination of structural domains of a target recognized by an antigen binding region of an antibody or antigen binding portion thereof. Epitopes can also consist of defined chemically active surface groupings of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural features as well as specific charge features. As used herein, the term "epitope" is defined as a portion of a polypeptide to which an antibody molecule can specifically bind, as determined by any method well known in the art, such as by conventional immunoassays, antibody competitive binding assays, or by x-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy).
The term "potency" is a measure of biological activity and may be designated as IC 50 、EC 50 Or an effective concentration of antibody or antibody drug conjugated to antigen CD122 that inhibits 50% of the activity as measured in the CD122 activity assay described herein.
As used herein in connection with the biological activity of an antibody disclosed herein, the term "inhibit" or "neutralizing" refers to the ability of an antibody to substantially antagonize, inhibit, prevent, limit, slow, destroy, eliminate, stop, reduce, or reverse the progression or severity of, for example, an inhibited activity, including, but not limited to, the biological activity or binding interaction of an antibody molecule with CD 122.
In this specification, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include any integer value within the range, and fractions thereof (such as tenths and hundredths of integers) where appropriate. The use of alternatives (e.g., "or") is understood to mean one, two, or any combination thereof. As used herein, the terms "include" and "comprise" are used synonymously.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of the documents define terms that contradict the definition of terms in the present application, the definition appearing in the present application controls. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein should not be taken as an admission or any form of suggestion that they form part of the effective prior art or form part of the common general knowledge in any country in the world.
Any of the aspects and embodiments described herein may be combined with any other aspects or embodiments disclosed herein in the summary, drawings, and/or detailed description, including the following specific non-limiting examples/embodiments of the disclosure.
Numbered embodiments
The disclosure sets forth the following numbered embodiments, albeit with the appended claims:
1. an anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
(a) The VH region amino acid sequence comprises the amino acid sequence of SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO:7 and the VL region amino acid sequence comprises an amino acid sequence comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15; or alternatively
(b) The VH region amino acid sequence comprises the amino acid sequence of SEQ ID NO:3, HCDR1 comprising SEQ ID NO:5 and HCDR2 comprising SEQ ID NO: HCDR3 of 7; and the VL region amino acid sequence comprises a sequence comprising SEQ ID NO:11, LCDR1 comprising SEQ ID NO:13 and LCDR2 comprising SEQ ID NO: LCDR3 of 15.
2. The antibody or antigen-binding portion of embodiment 1, wherein
(a) The VH region amino acid sequence comprises SEQ ID NO:1, and the VL region amino acid sequence comprises SEQ ID NO:17; or alternatively
(b) The VH region amino acid sequence comprises SEQ ID NO:1, and the VL region amino acid sequence comprises SEQ ID NO:9.
3. the antibody or antigen-binding portion of embodiment 1 or 2, wherein the antibody or antigen-binding portion is humanized or chimeric.
4. The antibody or antigen-binding portion of any one of embodiments 1-3, wherein the VH region, the VL region, or both the VH region and the VL region comprise one or more human framework region amino acid sequences.
5. The antibody or antigen-binding portion of any one of embodiments 1-4, wherein the VH region, the VL region, or both the VH region and the VL region comprise a human variable region framework scaffold amino acid sequence into which a CDR amino acid sequence has been inserted.
6. The antibody or antigen-binding portion of any one of embodiments 1 and 3-5, wherein the VH region comprises an IGHV3-23 human germline scaffold amino acid sequence into which HCDR1, HCDR2, and HCDR3 amino acid sequences have been inserted.
7. The antibody or antigen-binding portion of any one of embodiments 1 and 3-6, wherein the VL region comprises an IGKV1-33 human germline scaffold amino acid sequence into which LCDR1, LCDR2, and LCDR3 amino acid sequences have been inserted.
8. The antibody or antigen-binding portion of any one of embodiments 1-7, wherein the antibody comprises an immunoglobulin constant region.
9. The antibody or antigen binding portion of embodiment 8, wherein the immunoglobulin constant region is IgG, igE, igM, igD, igA or IgY.
10. The antibody or antigen-binding portion of embodiment 9, wherein the immunoglobulin constant region is IgG1, igG2, igG3, igG4, igA1, or IgA2.
11. The antibody or antigen-binding portion of embodiment 8, wherein the immunoglobulin constant region is immunologically inert.
12. The antibody or antigen-binding portion of embodiment 8, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitutions S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A, L235A and G237A, or a wild-type human IgG2 constant region, wherein numbering is according to the EU index as in Kabat.
13. The antibody or antigen-binding portion of embodiment 8, wherein the immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 32-38.
14. The antibody or antigen-binding portion of any one of embodiments 1-13, wherein the antibody or antigen-binding portion is Fab, fab ', F (ab') 2 Fv, scFv, macroantibody, minibody, diabody, triabody, tetrabody or diabody.
15. The antibody or antigen-binding portion of any one of embodiments 1-14, wherein the antibody is monoclonal.
16. The antibody or antigen-binding portion of any one of embodiments 1-15, wherein the antibody is a tetrameric antibody, a tetravalent antibody, or a multispecific antibody.
17. The antibody or antigen-binding portion of any one of embodiments 1-16, wherein the antibody is a bispecific antibody that specifically binds a first antigen and a second antigen, wherein the first antigen is CD122 and the second antigen is not CD122.
18. An immunoconjugate comprising the antibody or antigen binding portion of any one of embodiments 1-17 linked to a therapeutic agent.
19. The immunoconjugate of embodiment 18, wherein the therapeutic agent is a cytotoxin, radioisotope, chemotherapeutic agent, immunomodulatory agent, cytostatic enzyme, cytolytic enzyme, therapeutic nucleic acid, anti-angiogenic agent, anti-proliferative agent, or pro-apoptotic agent.
20. A pharmaceutical composition comprising the antibody or antigen-binding portion of any one of embodiments 1-17 or the immunoconjugate of embodiments 18 or 19, and a pharmaceutically acceptable carrier, diluent, or excipient.
21. A nucleic acid molecule encoding the antibody or antigen binding portion of any one of embodiments 1-17
(a) VH region amino acid sequence;
(b) VL region amino acid sequence; or (b)
(c) VH region amino acid sequence and VL region amino acid sequence.
22. An expression vector comprising the nucleic acid molecule of embodiment 21.
23. A recombinant host cell comprising the nucleic acid molecule of embodiment 21 or the expression vector of embodiment 22.
24. A method of producing an anti-CD 122 antibody, or antigen-binding portion thereof, comprising: culturing a recombinant host cell comprising the expression vector of embodiment 22 under conditions to express the nucleic acid molecule, thereby producing the antibody or antigen-binding portion; and isolating the antibody or antigen binding portion from the host cell or culture.
25. A method for inhibiting an immune response in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding portion of any one of embodiments 1-17, the immunoconjugate of embodiments 18 or 19, or the pharmaceutical composition of embodiment 20.
26. The method of embodiment 25, wherein the immune response is mediated by CD 122.
27. A method for treating or preventing a disease in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding portion of any one of embodiments 1-17, the immunoconjugate of embodiments 18 or 19, or the pharmaceutical composition of embodiment 20.
28. The method of embodiment 27, wherein the disease is an inflammatory disease or an autoimmune disease.
29. The method of embodiment 27, wherein the disease is vitiligo, celiac disease, type 1 diabetes, multiple sclerosis, graft versus host disease, systemic lupus erythematosus, psoriasis, atopic dermatitis, alopecia areata, ulcerative colitis, or rheumatoid arthritis.
30. A method for inhibiting IL-15-induced T cell migration from skin, the method comprising contacting the skin with a therapeutically effective amount of an antibody or antigen binding portion of any one of embodiments 1-17, an immunoconjugate of embodiment 18 or 19, or a pharmaceutical composition of embodiment 20.
31. The antibody or antigen-binding portion of any one of embodiments 1-17, the immunoconjugate of embodiments 18 or 19, or the pharmaceutical composition of embodiment 20, for use as a medicament.
The present disclosure will be further elucidated by the following examples, which are intended to be merely illustrative of the invention, and not limiting.
Examples
Example 1 optimized Generation of therapeutic antibodies against CD122
Introduction to the invention
In this example, we successfully generated a panel of antagonistic optimized anti-CD 122 antibodies. These anti-CD 122 antibodies are well expressed, biophysically stable, highly soluble, and have the greatest identity to the preferred human germline.
Materials and methods
Antibody v-domain specificity test: human receptor array analysis
Human cell membrane receptor proteome arrays were performed at Retrogenix Ltd.Primary screening: 5 μg/ml of IgG1-mikβ1 (humanized, also known as VillMab-1) antibodies were screened for binding to immobilized HEK293 cells/slides, which individually expressed 4975 human plasma membrane proteins (14 slide groups, n=2 slides per slide group). All transfection efficiencies exceeded the minimum threshold. Antibody binding was detected using AF647 fluorescent secondary anti-human IgG1 antibody. Primary hits (duplicate spots) were identified by analysis of fluorescence (AF 647 and ZsGreen 1) on ImageQuant. Vectors encoding all hits were sequenced to confirm their correct identity.
Confirmation/specificity screening: the vector encoding all hits plus the control vector encoding MS4A1 (CD 20) and EGFR were spotted on new slides and used to reverse transfect human HEK293 cells as previously described. All transfection efficiencies exceeded the minimum threshold. The same fixed slides were treated with 5 μg/ml of each test antibody, 5 μg/ml of negative control antibody, 1 μg/ml of rituximab anti-biomimetic drug (positive control), isotype IgG1 (Ab 00102 human IgG1 anti-fluorescein) or no test molecule (secondary antibody only; negative control) (n=2 slides per treatment). Slides were analyzed as described above.
Flow cytometry validation screening: expression vectors encoding ZsGreen1 alone or ZsGreen1 and CD122, BCAM were transfected into human HEK293 cells. Each live transfectant was incubated with 1 and 5mg/ml of each test antibody and isotype control antibody. Cells were washed and incubated with the same AF647 anti-human IgG Fc detection antibody as used in cell microarray screening. The cells were again washed and analyzed by flow cytometry using an Accuri flow cytometer (BD). Dead cells were excluded using 7AAD live/dead dye and ZsGreen1 positive cells (i.e., transfected cells) were selected for analysis.
Generation and selection of CD122 library
CD122 Fab libraries were assembled by large scale oligonucleotide synthesis and PCR. The amplified Fab library was then cloned into a phagemid vector by restriction ligation, transformed into E.coli TG-1 cells, and the phage library was rescued essentially as described in detail previously (Finlay et al 2011,Methods Mol Biol 681:383-401). Phage selection was performed by coating streptavidin magnetic microbeads with biotinylated CD122 target protein (human or cynomolgus monkey), washing the beads three times with PBS, and re-suspending in PBS ph7.4 plus 5% skim milk protein. In round 1 selection, these beads were coated with 100nM target protein, followed by a decrease in antigen concentration in successive rounds. In each round, phage were eluted with trypsin and subsequently re-infected into TG1 cells.
Expression and purification of Fab and IgG
Mammalian codon-optimized synthetic genes encoding the heavy and light chain variable regions of the leader anti-CD 122 antibodies and mikβ1 variants were cloned into mammalian expression vectors containing functionally null human IgG1 ("IgG 1-3M"; human IgG1 containing the L234A, L235A, G237A mutation in the lower hinge, which abrogates normal immunoglobulin ADCC, ADCP and CDC functions) and human ck domains, respectively. Vectors containing heavy and light chains were co-transfected in mammalian expression systems, followed by purification of IgG based on protein a, quantification and QC on denaturing and non-denaturing SDS-PAGE.
Direct binding ELISA for Fab and IgG
The binding and cross-reactivity of the leader group to the recombinant protein was initially assessed by binding ELISA. Coating human CD122 human Fc labeled recombinant protein and cynomolgus monkey and/or rhesus CD122 human Fc labeled recombinant protein at 1 μg/ml to MaxiSorp TM A surface of a flat bottom 96-well plate. Purified Fab or IgG samples were titrated in double serial dilutions starting from 500nM to 0.98nM and allowed to bind to the coated antigen. Fab was detected using mouse anti-c-myc antibody followed by donkey anti-mouse IgG conjugated to horseradish peroxidase. IgG was detected using mouse anti-human IgG conjugated to horseradish peroxidase. The binding signal was visualized with 3,3', 5' -tetramethylbenzidine substrate solution (TMB) and absorbance was measured at 450 nm.
Fab affinity to human and rhesus CD122Analysis
By at least one of3000 (GE) the affinity (KD) of purified IgG was determined by SPR on the solution with antigen. Following the two channel guide instructions, the use of amine coupling to fix mouse anti-human antibodies (CH 1 specificity) on CM5 sensor chip, in pH 4.5 acetate buffer to 2000RU levels. One channel is used for background signal correction. Standard running buffer HBS-EP pH 7.4 was used. Regeneration was performed by a single injection of 10. Mu.l of 10mM glycine (pH 1.5) at a rate of 20. Mu.l/min. 50nM of IgG sample was injected at 30. Mu.L/min for 2 min and then stopped for 60 seconds. Monomeric antigen (human CD122 His-tagged or cynomolgus monkey and/or rhesus CD122 His-tagged antigen) was reduced from 100nM to 3.1nM at twice serial dilutions, injected at 30 μl/min for 2 min, followed by a stop for 300 seconds. Use- >3000 evaluation (BIAevaluation) software analyzes the obtained sensorgram. KD was calculated by fitting to a 1:1 Langmuir binding model when association and dissociation were identical.
Flow cytometry of IgG
Purified IgG was tested for binding to human and rhesus CD122 expressed on CHO-K1 stable cell lines and CHO-K1 wild type cells in FACs. IgG samples were titrated in triplicate serial dilutions starting from 500nM to 0.98 nM. IgG binding was detected with FITC conjugated mouse anti-human IgG. The results were analyzed by examining the Mean Fluorescence Intensity (MFI) of 10000 cells per sample in a flow cytometer BL-1 channel detector (AttuneTM NxT Acoustic Focusing Cytometer, invitrogen/ThermoFisher Scientific).
M07e cell-based assays
M07e cells were obtained from DSMZ-German collection of microorganisms and cell cultures and maintained under the guidelines provided by the dealer at a concentration of 10% FBS, 10ng/mL GM-CSFPeprotech) And L-glutamine (Corning). On the day 1 of the day, the time period,cells were washed in RPMI and incubated at 2.5X10 5 The density of individual cells/mL was resuspended in RPMI supplemented with 10% fbs and L-glutamine (Corning). Recombinant human IL15 (rhIL 15) (R) at 50ng/mL&D) Or rhIL15 with antibody, in 96-well flat bottom plate wells, at 37 ℃ to a final volume of 200. Mu.L of total 5.0X10 4 Each cell lasted 72 hours. After 72 hours, cells were incubated with 20. Mu.L of WST-1 cell proliferation reagent (Miltenyi) for 3 hours at 37 ℃. Quantification of cell proliferation was performed with a scanning multi-well spectrophotometer and absorbance measured at 450nM was correlated with the number of living cells.
Human NK cell based assays
Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood of the donor using density gradient centrifugation at Ficoll Histopaque and NK cells were enriched from the isolated PBMCs using a Miltenyi Biotec (Bergisch Gladbach, germany) human NK cell isolation kit according to the manufacturer's instructions. According to the manufacturer's instructions, cellTrace is used TM CFSE cell proliferation kit NK cells were stained and resuspended in RPMI supplemented with 10% fbs and penicillin-streptomycin (Gibco). Recombinant human IL15 (rhIL 15) (R) at 20ng/mL&D) Or rhIL15 with antibody in the presence of 96-well round bottom plate wells, at 37 ℃ for a total of 10 culture 5 Individual NK cells lasted 120 hours. After 120 hours, NK cells were washed and stained with anti-human CD3 (UCHT 1), CD56 (5.1H11), CD16 (3G 8) (1:20 dilution, bioleged) and CFSE dilutions were analyzed with BD LSR II flow cytometry (BD Biosciences) and FlowJo (Tree Star inc.).
NSG-IL15 mouse model
Humanized mice were generated by transplanting NOD scid mice expressing human IL15 (NSG-Tg) with human Hematopoietic Stem Cells (HSCs). At the time of injection 10 derived from cord blood 5 Prior to cd34+ HSCs, NSG-Tg mice of 6 to 8 weeks of age received 200cGy of radiation. HSC transplanted NSG-Tg mice were screened at 12 and 16 weeks to determine baseline transplantation in blood. NSG-Tg mice with more than 20% of human cd45+ cells, i.e. more than 2% cd56+, were selected for antibody treatment. Mice were treated with intraperitoneal (i.p.) injection twice weekly (monday/thursday schedule) for duration3 weeks. The human immune cell levels in the blood were quantified using flow cytometry at 1 week and 3 weeks after initiation of treatment and at 1 week, 3 weeks and 5 weeks after treatment. Mice were euthanized 5 weeks after treatment and human immune cell levels in spleen and blood were measured by flow cytometry. Cells from all tissues were stained with anti-human CD45, CD3, CD4, CD8, CD7, CD56, CD16, mik-b2 and Mik-b3 (1:20 dilution, biolegend) and analyzed with BD LSR II flow cytometer (BD Biosciences) and FlowJo (Tree Star Inc.).
Results and discussion
Pharmacological model: assessing the feasibility of IL15 Rbeta antagonist antibodies for use in the treatment of skin disorders
Computer modeling was performed to determine pharmacological parameters and characteristics that might drive the success of anti-CD 122 IgG1 antibody treatment designed to maximize efficacy in specific tissues such as skin. These assays were based on the established values of CD122 target biology (table 1) and known potential drug properties and dose parameters of bivalent IgG antibodies (table 2).
The above parameters were then analyzed to sample their effect on drug efficacy and distribution as shown in figure 1. The bottom-up estimation method was confirmed using PK data of reported t-cell leukemia patients previously dosed with the humanized anti-CD 122 IgG1 antibody 'mikβ1'. These assays resulted in a range of findings related to IV and/or SC dosing and antibody CD122 binding affinity in average body weight human subjects:
1. 700mg IV Q4W or 100mg SC Q1W was expected to consistently inhibit IL15Rβ (> 90%) in skin at a nominal drug affinity of 10nM KD (tables 3, 4).
2. Higher functional affinity than 10nM KD can reduce dose requirements for IV and SC, with KD values closer to 1nM, allowing 99% target occupancy in skin at maximum dose. This pattern was qualitatively similar for IV and SC routes of administration (tables 3, 4).
3. 100mg of SC Q1W is sufficient to maintain 90% receptor occupancy in the skin, but 100mg of SC Q1W is insufficient to maintain > 95% receptor occupancy at a nominal drug binding affinity of 10 nM. In the 100mg SC q1w dosing regimen, higher affinities were required to achieve > 95% receptor occupancy (table 4).
4. Elimination of systemic target uptake has minimal effect on the dose required to achieve high (> 90%) target occupancy in the skin. Simulations of IV Q4W dosing in the presence of known levels of systemic CD122 compared to the absence of CD122 indicate that the total dose must overcome systemic target loading to have optimal activity in the skin and drug profile effects (table 5).
Taken together, these assays demonstrate that the optimal anti-CD 122 antagonistic therapeutic antibody has a functional affinity for CD122 of > 10nM, allowing administration at 700mg IV q4w or 100mg SC q1 w. The increased affinity allows for greater target coverage at lower doses.
Antibody binding specificity assay
In early clinical trials, the humanized anti-CD 122 IgG1 antibody "mikβ1" was reported to exhibit evidence of accelerated clearance. Accelerated clearance is a risk factor that cannot achieve the desirable drug characteristics described above, as the target-mediated drug profile (TMDD) effect can negatively impact drug efficacy. It is hypothesized that mikβ1 may bind not only CD122, but also unknown and unpredictable human proteins. To examine this possibility, off-target binding specificity in humanized IgG1-mikβ1 (VillMab-1) was screened using in vitro techniques (Retrogenix, ltd.) based on the use of high density cell arrays expressing > 5500 unique human membrane receptors and membrane tethered secretion proteins. This receptor array binding screen established that VillMab-1 showed strong binding to membrane-expressed CD122, but also had potential off-target binding specificities for BCAM (also known as AU, CD239, LU, MSK19, basal cell adhesion molecule (luthan blood group)). BCAM is a widely expressed membrane adhesion protein that can lead to PK lowering and exacerbate the "absorbing" effect in therapeutic administration of anti-CD 122 antibodies.
To confirm this off-target binding event, DNA sequencing was performed on plasmids encoding BCAM and control proteins. These analyses confirm that the encoded protein is indeed the correct sequence. Plasmid samples of control and potential target receptors were then rearranged onto a new chip for duplicate analysis. By scanning ZS green in the chip, efficient induction of expression of all rearranged plasmids was confirmed, the genes being co-encoded as internal control markers on all expression plasmids. This analysis showed that ZS expression was clearly detected at all positions of the spotted plasmid (fig. 2A). In addition, transfected cells were then re-probed with VillMab-1 (fig. 2B), rituximab (IgG 1 positive control, fig. 2C) and a chip without the application of a primary antibody probe (fig. 2D) using the same spotted slide. These analyses showed that VillMab-1 again showed measurable binding above background (on both chips) on cells transfected with BCAM (fig. 2B). As expected, rituximab demonstrated binding to CD20, and no binding to any other protein was observed (fig. 2C). In chips not probed with primary antibodies (fig. 2D), only the expected control protein showed any signal. This clean performance of the control chip confirms that the VillMab-1 binding signal on CD122 and BCAM is specific. To further confirm the findings of these off-target binding, sequence-verified plasmids were re-transfected into HEK-293 cells and binding was studied by flow cytometry (fig. 3). In this experiment, villMab-1 showed clear binding to both CD122 and BCAM transfected cells, but no background binding to cells transfected with ZS ("ZS only", fig. 3A). Control experiments using the same cells but without primary antibodies (fig. 3B) indicated that BCAM signals were antibody-related and staining with rituximab IgG1 against CD20 (fig. 3C) showed only binding signals on CD20 transfected cells, demonstrating that the signals on BCAM are mediated specifically by the binding domain of VillMab-1.
Therapeutic antibodies should ideally be highly specific for their desired targets, as off-target binding has been shown to have potentially negative effects on IgG PK, biodistribution and toxicity characteristics. To solve this problem of VillMab-1, exploratory modulation of the VillMab-1 binding interface was performed as follows.
VillMab-1 mutagenesis and paratope modulation
In order to bias engineering efforts towards final lead therapeutic IgG compounds with optimal drug-like properties, variants were selected to examine the mutagenic origin of the VillMab-1 antibodies. Sequence analysis of the v domain of VillMab-1 showed that the initial humanization process used scaffolds associated with the human germline frameworks IGHV3-23 and IGKV1-33, which were known to have good solubility and drug development quality and were used at high frequency in the expressed human antibody repertoire (table 6). Although well-known scaffolds are used, the framework of the variable domain contains a large number of deviations from germline sequences. In addition, the CDR sequences also contain a number of residues which differ from the human germline (table 6).
V-domain sequences of VillMab-1 were combined into Fab phage display format and independent mutagenesis library cassettes for VH and VL domains were generated by oligomer synthesis and assembly. Each mutagenesis cassette encodes a VillMab-1 residue, a human germline residue or a homologous amino acid at each underlined position in table 6. The mutation cassette of VL was combined with the mutagenesis cassette of VillMab-1 VH or VillMab-1 VL with VH to generate separate Fab libraries. Each final Fab library was ligated into phage display vector and transformed into E.coli by electroporation to yield > 10 7 Individual clones. Library construction quality was verified by sequencing 96 clones per library. The sequencing data shows that the location of the mutation effectively samples the diversity of the design. Helper phage M13 was used to rescue the library and select for human and cynomolgus monkey and/or rhesus monkey CD122-Fc proteins biotinylated in multiple separate branches. After round 1 selection, a third combinatorial library was created using the preselected mutated VH and VL combinations, which simultaneously sampled the variability of selection in both V domains.
Periplasmic preparation screening and DNA sequencing after selection revealed the presence of 64 unique human and rhesus CD122 binding Fab clones that showed strong binding to human and rhesus CD122 in ELISA and > 50% inhibition of VillMab-1 binding to human and rhesus CD122 in the Alphascreen assay. From these unique, library-derived leader sequences, the first 15 clones were identified based on the intensity of the measured signal, the level of mutation to the human germline and the lack of major developmental propensity/chemical degradation motifs (table 7). This analysis also identified a unique series of sequences in each CDR (table 8). These unique CDR sequence profiles were used to design 15 other clones (MAB 01-MAB 15) with potential CDR combinations not found in the first 15 library-derived clones, as shown (table 9). These 30 total unique clones were expressed in CHO transient cultures as human IgG1, purified by protein a, and confirmed by size exclusion chromatography to be > 95% monomeric.
Leader IgG specificity and efficacy profile
The purified IgG was then tested in the alpha screen format for competition for VillMAb-1 binding epitope on human CD 122-Fc. This analysis showed that 24 of the 30 clones reduced binding of VillMab-1 to CD122 in a concentration-dependent manner, demonstrating that they retained the functional epitope of the parent antibody (fig. 4). These 24 clones were then examined in a multi-reactivity assay to ensure that the final lead clone from the initial engineering did not have DNA or insulin binding spectra strongly correlated with short PKs (fig. 5). This analysis showed that while all 24 clones showed significantly lower signals than the positive control primary coomassie mab (Bococizumab), the subset generated particularly low signals equivalent to or better than the negative control antibodies Bevacizumab, motekumab (usekin umab) and Pembrolizumab (fig. 5). These findings allow for prioritization of 6 key cues for further analysis.
The concentration-dependent binding of 6 prioritized library-derived precursor clones to human and rhesus CD122 on the cell surface was analyzed by flow cytometry (fig. 6). Clone 06F11 (fig. 6A), 07C07 (fig. 6B), 07D06 (fig. 6C), 07E09 (fig. 6D), 07D07 (fig. 6E), and 06D12 (fig. 6F) each exhibited a CD 122-specific binding profile that was highly similar to the profile observed for VillMab-1, whereas isotype control IgG1 did not exhibit binding to any cell type (fig. 6G).
Leading IgG analysis in CD122-IL-15 blocking assay
In the M07E cell-based CD122/IL-15 blocking reporter assay, clones 06F11 (fig. 7A), 07C07 (fig. 7B), 07D06 (fig. 7C), 07E09 (fig. 7D), 07D07 (fig. 7E), and 06D12 (fig. 7F) exhibited concentration-dependent antagonism of CD 122. The IC50 of VillMAB-1 was 9.792. Mu.g/mL. IC50 s of 6F11 and 7C07 were 14.8. Mu.g/mL and 20.8. Mu.g/mL, respectively, and were the lowest of the pilot clones. IC 50's for 06D12, 07D06, 07D07 and 07E09 were 38.610 μg/mL, 27.820 μg/mL, 34.170 μg/mL and 23.610 μg/mL, respectively. This analysis emphasizes 06F11 and 07C07 as ideal candidates for further evaluation.
CD122 binding to 1:1 binding affinityLeading Fab analysis of (C)
To characterize clones 06F11 and 07C07 plus the true 1:1 affinity value of the 06F11 variant corrected for FW1 (06F 11-V) mutations, these clones were cloned, expressed and purified in human Fab format and the positive control Villmab-1 clone (i.e., monovalent and lacking both hinge and Fc regions). The binding of fully purified Fab proteins to both human CD122 (table 10) and rhesus CD122 (table 11) was examined. These analyses showed that clones FAB06F11-V, FAB06F11 and FAB07C07 showed moderately reduced overall KD values for human and rhesus CD122 compared to VillFab-1, but importantly, both also showed increased association rate (ka) and dissociation rate (KD).
Lead IgG analysis in hIL-15NSG mouse model
The humanized hIL-15NSG mouse model was used to compare the ability of VillMAB-1, 7C07 and 6F11-v (all in the IgG1-3M effector-null form) to inhibit human NK and CD 8T cell transplantation supported by CD122/IL15 signaling in vivo. After complete transplantation with cells was established, mice were treated with vehicle, low dose (1 mg/kg) or high dose (10 mg/kg) of antibody.
The number of human cd8+ T cells (fig. 8A) and NK cells (fig. 8B) in the blood between groups of mice was comparable prior to antibody treatment. After 1 week of treatment, mice treated with only 10mg/kg VillMAB-1 showed a statistically significant decrease in cd8+ T cell numbers compared to mice treated with the isotype (fig. 8C). Cd8+ T cell depletion was observed for all antibody treated groups when compared to mice treated with isotype. The number of NK cells in the blood was comparable in all groups (fig. 8D).
After 3 weeks of treatment, the number of cd8+ T cells in the blood was reduced in all antibody-treated groups compared to isotype-treated mice (fig. 8E). These changes are not statistically significant. The number of NK cells was reduced in all groups of antibody-treated mice, and this reduction was statistically significant in each group except mice treated with 1mg/kg07C07 (fig. 8F). High and low doses of VillMAB-1 treatment reduced the number of NK cells more than either dose of 06F11-V or 07C07 treatment.
Importantly, these findings demonstrate that chronic blockade of CD122-IL15 signaling by clone Villmab-1, 06F11-V or 07C07 in the absence of ADCC or ADCP effector function has the ability to drive depletion of NK and cd8+ t cell populations.
Antibody binding specificity analysis of clones 06F11-V and 07C07
Since clones 06F11-V and 07C07 were shown to exhibit a high level of humanization in the framework regions and CDRs of the V-domain above, coupled with effective blocking of CD122 in vitro and in vivo, they were used for rescreening for specific analysis on a Retrogenix proteomics platform. Unexpectedly, this analysis showed that the BCAM binding observed for Villmab-1 had been completely removed, whereas no binding was observed for either of 06F11-V and 07C07 IgG. However, in addition, two new interactions were observed, which were not observed for Villmab-1 (fig. 9). In flow cytometry analysis of binding to cells transfected with plasmids driving target and control expression, clone 06F11-V was found to bind to neudins (a neurotrophin) and CILP2 (a cartilage structural protein) in addition to CD122 binding, while 07C07 was found to bind only to CILP2 (fig. 9A, 9B). In contrast, "secondary antibody only" (fig. 9C) and rituximab primary antibody (fig. 9D) control experiments demonstrated no binding above background, or binding only to CD20 transfected cells, respectively. These findings confirm that off-target binding of 06F11-V and 07C07 IgG is authentic and specific.
Optimization of clone 06F11-V
Clone 06F11-V was selected for further optimization to maximize beneficial properties and minimize off-target binding. This optimization was performed by experimental analysis of combinations of mutations back to murine sequences in CDR1, and/or CDR2, and/or CDR3 of the heavy and light chain sequences of 06F-11V. This procedure resulted in the generation of 18 new clones, each carrying a combination of one of the 6 VH sequences with one of the 3 VL sequences (table 12). These 18 novel variants, villmab-1 and 06F11V, were cloned as human monovalent Fab fragments, expressed in CHO cells, purified to monomer state by protein a column and then SEC.
Then pass throughThe purified fab was examined for binding affinity to human and rhesus CD122 (table 13). This analysis shows that a series of clones showed an affinity for CD122 better than 06F11-V and even better than VillMab-1. From this group, clones MAB05, MAB06, MAB14, MAB15, MAB17 and MAB18 were preferentially used for further analysis and were further expressed and purified in the form of human IgG1-3M for potency and specificity analysis. When in->When the Fab versions of all prioritized clones were tested in binding to human neudins and CILP2 proteins (fig. 10A, 10B), it was found that clone 06F11-V alone showed measurable binding to either protein, indicating that the novel leader clone eliminated the risk of off-target binding of both. In ELISA analysis of IgG from all clones on human BCAM protein, all clones except MAB05 and MAB06 retained off-target binding, which showed complete specificity for CD122 (fig. 10C, table 14).
To examine how it is possible that such closely related antibody sequences have such fundamental differences in specificity profile, sequence alignments were performed for VH and VL domains of VillMab-1 with clones 05, 06, 14, 15, 17 and 18 (fig. 11A, 11B). Notably, the only changes specific for (complete CD 122-specific) clones MAB05 and MAB06, other than VillMab-1 sequences, were the 3 (highly homologous) mutations found in and near CDR1 of the VH domain. This finding demonstrates the unpredictable nature of the confusion of antibody binding.
Confirmation of the biological titers of clones MAB05, MAB06, MAB14, MAB15, MAB17 and MAB18 was determined in the IL-15 stimulated M07e assay (Table 14). This analysis showed that not only did the affinity of clones MAB05 and MAB06 increase compared to VillMab-1, but they also increased approximately twice as much in potency to block IL-15 signaling (Table 14). The final functionally relevant efficacy of clones MAB05 and MAB06 was then determined in an assay that measures proliferation of human primary NK cells under IL-15 stimulation (FIG. 12A, FIG. 12B). This assay summarises the findings in the M07e assay, demonstrating that MAB05 and MAB06 are indeed improved compared to VillMab-1.
IgG1-3M binding to human and murine Fc receptors Analysis
To characterize the affinity of clone 06F11-V IgG1-3M and positive control IgG, the binding of the fully purified protein to human and murine fcγr and FcRn was examined (table 17). These analyses showed that clone 06F11-V IgG1-3M showed very low or no measurable affinity for human or murine fcγr and FcRn at pH 7.4, although all positive controls showed the expected strong interactions with human and murine receptors. Importantly, however, 06F11-V IgG1-3M maintained full affinity for human and murine FcRn at pH 6.0. These findings confirm that the above-described in vivo observations in the NSG/IL-15 mouse model, i.e., blocking CD122 signaling in the absence of IgG effector function, is sufficient to deplete CD122+ cells.
Example 2 anti-CD 122 therapeutic antibody function in a human skin T cell climbing assay
Human skin biopsy culture assays were used to assess the ability of MAB05 and MAB06 to inhibit CD122/IL-15 signaling in skin resident T cells.
Human skin biopsies (4 mm diameter x 2mm thickness) were collected from surgical specimens (lipoteichiometrics) using a 4mm integrate disposable biopsy punch (integrate). Skin biopsies were incubated at 4 ℃ for 30 minutes in antibiotic-antifungal (Gibco) diluted with PBS, then rinsed 3 times with PBS. Three skin biopsies were placed in 1 well of a 24 well plate (Corning) and briefly dried to promote adhesion of the biopsies to the well surface. The biopsies were then cultured in 2mL Iscove's modified medium (Sigma) with 20% heat-inactivated fetal bovine serum, penicillin and streptomycin (Corning) and 3.5. Mu.L/L beta-mercaptoethanol (Sigma) and incubated at 37℃for 21 days. Cultures were fed three times per week by pipetting 1mL of medium from each well and adding back 1mL of fresh medium. For cultures treated with IL-15 or anti-CD 122 antibodies, 20ng/mL recombinant human IL15 (rhIL 15) (R & D) and anti-CD 122 antibodies (MAB 05 or MAB 06) were added from the beginning of the culture until T cells were harvested on day 21. After 21 days, the medium was harvested from the wells and centrifuged at 330 Xg for 10 minutes in a 5mL polystyrene round bottom tube. The supernatant was aspirated, the remaining T cells were washed, stained with anti-human CD3, CD4 and CD8 (1:20 dilution, biolegend), and quantified with BD LSR II flow cytometry (BD Biosciences) and FlowJo (Tree Star inc.).
In this assay, MAB05 and MAB06 inhibited IL 15-induced accumulation of CD8+ T cells migrating from skin biopsies. More cd8+ T cells accumulated when the biopsies were incubated with IL-15 than when the biopsies were not incubated with IL-15; 11, 101.+ -. 6011 vs 438.3.+ -. 66.05 (mean.+ -. SD) (FIG. 13A). When biopsies are incubated with IL-15 and MAB05, the number of accumulated CD8+ T cells is reduced compared to incubation with IL-15 alone; 2096.+ -. 1100 vs. 11, 101.+ -. 6011 (mean.+ -. SD) (FIG. 13A). Similarly, when biopsies were incubated with IL-15 and MAB06, fewer CD8+ T cells accumulated than with IL-15 alone; 2436.+ -. 501.6 compared to 11, 101.+ -. 6011 (mean.+ -. SD) (FIG. 13A).
MAB05 and MAB06 also inhibited IL 15-induced accumulation of CD4+ T cells that migrated from skin biopsies. More CD4+ T cells accumulated when the biopsies were incubated with IL-15 than when the biopsies were not incubated with IL-15; 40,523.+ -. 15,391 versus 1261.+ -. 473.6 (mean.+ -. SD) (FIG. 13B). When biopsies are incubated with IL-15 and MAB05, the number of accumulated CD4+ T cells is reduced compared to incubation with IL-15 alone; 3471.+ -. 1627 vs 40,523.+ -. 15391 (average.+ -. SD) (FIG. 13B). Similarly, when biopsies were incubated with IL-15 and MAB06, fewer CD4+ T cells accumulated compared to incubation with IL-15 alone; 4308+ -2111 vs 40,523 + -15391 (average+ -SD) (FIG. 13B).
MAB05 showed concentration-dependent antagonism against IL-15-induced accumulation of skin resident CD8+ T cells in the skin biopsy culture assay with an IC50 of 1.9 μg/mL (FIG. 14A). MAB06 showed comparable concentration-dependent antagonism of CD8+ T cell accumulation with an IC50 of 1.8 μg/mL (FIG. 14A). In addition, MAB05 and MAB06 showed considerable concentration-dependent antagonism of IL-15 induced accumulation of CD4+ T cells with IC50 of 2.1 μg/mL and 1.8 μg/mL, respectively (FIG. 14B).
Example 3 pharmacokinetic/pharmacodynamic (PK/PD) studies of cynomolgus monkey anti-CD 122 therapeutic antibodies
A single intravenous infusion of anti-CD 122 antibody (MAB 05 or MAB 06) was administered to cynomolgus monkeys at a dose level ranging from 1 to 20mg/kg. Blood samples were collected at different time points before and 1 hour after dosing to day 16 post dosing. Pharmacokinetic parameters were determined after quantitation of anti-CD 122 antibody plasma concentrations by ELISA methods. Blood samples were also analyzed for CD122 receptor occupancy on NK cells and quantification of total T cells, helper T cells, cytotoxic T cells and NK cells using flow cytometry methods.
MAB05 and MAB06 showed no clinical abnormalities and linear pharmacokinetics in cynomolgus monkeys after a single intravenous administration at a dose level of 1mg/kg to 20mg/kg. A single dose of 1mg/kg MAB05 and MAB06 was sufficient to maintain > 90% CD122 receptor occupancy on NK cells throughout the sampling period (16 days post-dosing). Administration of single doses of 1mg/kg, 5mg/kg or 20mg/kg of MAB05 and MAB06 induced a reduction in circulating NK cell numbers. Approximately 7 days after dosing, circulating NK cells reached a minimum and showed stable recovery after a dose of 1mg/kg or 5mg/kg during the remaining sampling period (16 days after dosing). Modulation of circulating NK cell numbers is considered a marker of functional activity and is critical to demonstrate the efficacy of anti-CD 122 antibodies in vivo (see Waldmann et al (2020) J Exp Med 217:e20191062) and can therefore be advantageously used to determine optimal doses for patients. No effect on helper T cells or cytotoxic T cells was observed.
EXAMPLE 4 Pre-formulation development and stability Studies of therapeutic antibodies against CD122
A set of stability studies was used to evaluate the production developability of anti-CD 122 antibodies. The stability and aggregation potential of ten anti-CD 122 antibody formulations at 5mg/mL concentration were evaluated from a combination of buffer and excipients. Antibodies were evaluated under low pH stress, heat stress, freeze thawing conditions, and forced oxidation conditions. Self-association and viscosity of the antibodies were also assessed at concentrations > 100 mg/mL.
MAB05 and MAB06 exhibited high thermal stability, high tolerance to low pH (pH 3.0), low aggregation potential, low oxidation and deamidation sensitivity and excellent freeze/thaw stability in buffers with a composition of 25mM L-histidine, 9% (w/v) sucrose, 0.02% (w/v) polysorbate 80 (pH 6.0). MAB05 and MAB06 were dissolved in this same buffer solution at concentrations of 90 to 120 mg/mL. The lower viscosity (approximately 4-6 centipoise) of MAB05 and MAB06 at 115mg/mL indicates the feasibility of clinical formulations to achieve subcutaneous delivery of anti-CD 122 antibodies.
TABLE 1 target biological input parameters for pharmacological modeling of anti-CD 122 IgG
TABLE 2 drug infusion parameters for pharmacological modeling of anti-CD 122 IgG
Iv q4w dosing: influence of affinity of drug for CD122 on dosing requirements (minimum dose required to meet inhibition criteria at different drug affinities)
Sc q1w dosing: SC administration requirement of 10nM KD
(the predicted dose of RO is reached in the skin, assuming 100mg is the maximum feasible SC dose)
Iv q4w: target RO dose without systemic burden (10 nM IgG KD)
TABLE 6 amino acid sequence of humanized anti-CD 122 variable region
Bold = CDR
Underlined = different from human germline IGHV3-23/JH4 (VH) and IGKV1-33/J4 (VL)
TABLE 7 variable region sequences of the first 15 unique library-derived CD122 antagonistic IgGs
Table 8. Unique CDR sequences found in library-derived CD122 binding antibodies.
TABLE 9 designer variable domain sequences used in pairwise combinations to form MAB01-MAB15 IgG1
TABLE 10 Fab binding to human CD122Affinity value
TABLE 11 Fab binding to rhesus CD122Affinity value
TABLE 15 examples of amino acid sequences of Fc regions of antibodies
Human IgG4 wild type
(SEQ ID NO：32)
Human IgG4 (S228P)
(SEQ ID NO：33)
Human IgG1 wild type
(SEQ ID NO：34)
Human IgG1-3M
(SEQ ID NO：35)
Human IgG2 wild type
(SEQ ID NO：36)
Human IgG1 wild type "REEM" allotype
(SEQ ID NO：37)
Human IgG1-3M "REEM" allotype
(SEQ ID NO：38)
TABLE 16 examples of amino acid sequences of membrane proteins
Human CD122 sequence
(SEQ ID NO：19)
Cynomolgus monkey and rhesus monkey CD122 sequences
(SEQ ID NO：20)
Human BCAM sequences
(SEQ ID NO：21)
TABLE 17 Fc receptor interactionAnalysis
TABLE 18 amino acid sequence of antibodies MAB05
CDR sequences in the variable region sequences are underlined.
TABLE 19 amino acid sequence of antibodies MAB06
CDR sequences in the variable region sequences are underlined.
TABLE 20 amino acid sequence of murine/humanized antibody MIK beta 1
CDR sequences in the variable region sequences are underlined.
Sequence listing
<110> verarism treatment Co., ltd (Villaris Therapeutics, inc.)
<120> anti-CD 122 antibody and use thereof
<130> VLRS-001/02WO 338855-2003
<150> US 63/279,762
<151> 2021-11-16
<150> US 63/174,772
<151> 2021-04-14
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<170> PatentIn version 3.5
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290 295 300
Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro
305 310 315 320
Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu
325 330 335
Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ser Ser Leu Ser Ser Asn
340 345 350
Arg Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His
355 360 365
Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr
370 375 380
Asp Pro Cys Ala Glu Glu Glu Pro Asp Glu Gly Gly Ala Asp Ala Pro
385 390 395 400
Thr Gly Ser Ser Pro Gln Pro Leu Arg Pro Leu Ser Ala Glu Asp Asp
405 410 415
Ala Tyr Cys Thr Phe Pro Ser Gly Asp Asp Leu Leu Leu Phe Ser Pro
420 425 430
Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser
435 440 445
Gly Ala Gly Glu Glu Arg Leu Pro Pro Ser Leu Gln Glu Arg Val Pro
450 455 460
Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro
465 470 475 480
Asp Leu Val Asp Phe Gln Pro Arg Pro Glu Leu Val Leu Arg Glu Ala
485 490 495
Gly Glu Gln Val Pro Asp Pro Gly Pro Arg Glu Pro Phe Ser Phe Pro
500 505 510
Trp Ala Arg Pro Pro Gly Gln Gly Glu Val Arg Ala Leu Asn Ala Arg
515 520 525
Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Asp
530 535 540
Gln Asp Pro Thr His Leu Val
545 550
<210> 21
<211> 628
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 21
Met Glu Pro Pro Asp Ala Pro Ala Gln Ala Arg Gly Ala Pro Arg Leu
1 5 10 15
Leu Leu Leu Ala Val Leu Leu Ala Ala His Pro Asp Ala Gln Ala Glu
20 25 30
Val Arg Leu Ser Val Pro Pro Leu Val Glu Val Met Arg Gly Lys Ser
35 40 45
Val Ile Leu Asp Cys Thr Pro Thr Gly Thr His Asp His Tyr Met Leu
50 55 60
Glu Trp Phe Leu Thr Asp Arg Ser Gly Ala Arg Pro Arg Leu Ala Ser
65 70 75 80
Ala Glu Met Gln Gly Ser Glu Leu Gln Val Thr Met His Asp Thr Arg
85 90 95
Gly Arg Ser Pro Pro Tyr Gln Leu Asp Ser Gln Gly Arg Leu Val Leu
100 105 110
Ala Glu Ala Gln Val Gly Asp Glu Arg Asp Tyr Val Cys Val Val Arg
115 120 125
Ala Gly Ala Ala Gly Thr Ala Glu Ala Thr Ala Arg Leu Asn Val Phe
130 135 140
Ala Lys Pro Glu Ala Thr Glu Val Ser Pro Asn Lys Gly Thr Leu Ser
145 150 155 160
Val Met Glu Asp Ser Ala Gln Glu Ile Ala Thr Cys Asn Ser Arg Asn
165 170 175
Gly Asn Pro Ala Pro Lys Ile Thr Trp Tyr Arg Asn Gly Gln Arg Leu
180 185 190
Glu Val Pro Val Glu Met Asn Pro Glu Gly Tyr Met Thr Ser Arg Thr
195 200 205
Val Arg Glu Ala Ser Gly Leu Leu Ser Leu Thr Ser Thr Leu Tyr Leu
210 215 220
Arg Leu Arg Lys Asp Asp Arg Asp Ala Ser Phe His Cys Ala Ala His
225 230 235 240
Tyr Ser Leu Pro Glu Gly Arg His Gly Arg Leu Asp Ser Pro Thr Phe
245 250 255
His Leu Thr Leu His Tyr Pro Thr Glu His Val Gln Phe Trp Val Gly
260 265 270
Ser Pro Ser Thr Pro Ala Gly Trp Val Arg Glu Gly Asp Thr Val Gln
275 280 285
Leu Leu Cys Arg Gly Asp Gly Ser Pro Ser Pro Glu Tyr Thr Leu Phe
290 295 300
Arg Leu Gln Asp Glu Gln Glu Glu Val Leu Asn Val Asn Leu Glu Gly
305 310 315 320
Asn Leu Thr Leu Glu Gly Val Thr Arg Gly Gln Ser Gly Thr Tyr Gly
325 330 335
Cys Arg Val Glu Asp Tyr Asp Ala Ala Asp Asp Val Gln Leu Ser Lys
340 345 350
Thr Leu Glu Leu Arg Val Ala Tyr Leu Asp Pro Leu Glu Leu Ser Glu
355 360 365
Gly Lys Val Leu Ser Leu Pro Leu Asn Ser Ser Ala Val Val Asn Cys
370 375 380
Ser Val His Gly Leu Pro Thr Pro Ala Leu Arg Trp Thr Lys Asp Ser
385 390 395 400
Thr Pro Leu Gly Asp Gly Pro Met Leu Ser Leu Ser Ser Ile Thr Phe
405 410 415
Asp Ser Asn Gly Thr Tyr Val Cys Glu Ala Ser Leu Pro Thr Val Pro
420 425 430
Val Leu Ser Arg Thr Gln Asn Phe Thr Leu Leu Val Gln Gly Ser Pro
435 440 445
Glu Leu Lys Thr Ala Glu Ile Glu Pro Lys Ala Asp Gly Ser Trp Arg
450 455 460
Glu Gly Asp Glu Val Thr Leu Ile Cys Ser Ala Arg Gly His Pro Asp
465 470 475 480
Pro Lys Leu Ser Trp Ser Gln Leu Gly Gly Ser Pro Ala Glu Pro Ile
485 490 495
Pro Gly Arg Gln Gly Trp Val Ser Ser Ser Leu Thr Leu Lys Val Thr
500 505 510
Ser Ala Leu Ser Arg Asp Gly Ile Ser Cys Glu Ala Ser Asn Pro His
515 520 525
Gly Asn Lys Arg His Val Phe His Phe Gly Thr Val Ser Pro Gln Thr
530 535 540
Ser Gln Ala Gly Val Ala Val Met Ala Val Ala Val Ser Val Gly Leu
545 550 555 560
Leu Leu Leu Val Val Ala Val Phe Tyr Cys Val Arg Arg Lys Gly Gly
565 570 575
Pro Cys Cys Arg Gln Arg Arg Glu Lys Gly Ala Pro Pro Pro Gly Glu
580 585 590
Pro Gly Leu Ser His Ser Gly Ser Glu Gln Pro Glu Gln Thr Gly Leu
595 600 605
Leu Met Gly Gly Ala Ser Gly Gly Ala Arg Gly Gly Ser Gly Gly Phe
610 615 620
Gly Asp Glu Cys
625
<210> 22
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain variable region
<400> 22
Glu Val Gln Leu Leu Glu 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 Ser Val Thr Ser Tyr
20 25 30
Gly Val His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Phe
65 70 75 80
Gln Met Asn Ser Leu Gln Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 23
<211> 30
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain FR1
<400> 23
Glu Val Gln Leu Leu Glu 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 Ser Val Thr
20 25 30
<210> 24
<211> 5
<212> PRT
<213> mice (Mus musculus)
<400> 24
Ser Tyr Gly Val His
1 5
<210> 25
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain FR2
<400> 25
Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly
1 5 10
<210> 26
<211> 32
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> heavy chain FR3
<400> 26
Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Phe Gln
1 5 10 15
Met Asn Ser Leu Gln Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg
20 25 30
<210> 27
<211> 11
<212> PRT
<213> mice (Mus musculus)
<400> 27
Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 28
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain variable region
<400> 28
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Gly Ser Ser Ser Val Ser Phe Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Tyr Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Val Lys
100 105
<210> 29
<211> 23
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain FR1
<400> 29
Asp Ile Val Leu 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> 30
<211> 10
<212> PRT
<213> mice (Mus musculus)
<400> 30
Ser Gly Ser Ser Ser Val Ser Phe Met Tyr
1 5 10
<210> 31
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> light chain FR4
<400> 31
Phe Gly Gln Gly Thr Lys Val Glu Val Lys
1 5 10
<210> 32
<211> 327
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 32
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser 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 Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 33
<211> 327
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IgG4 (S228P)
<400> 33
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser 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 Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 34
<211> 330
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 34
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 Gly Lys
325 330
<210> 35
<211> 330
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> IgG1-3M
<400> 35
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 Ala 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 Gly Lys
325 330
<210> 36
<211> 326
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 36
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser 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 Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 37
<211> 330
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 37
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 Gly Lys
325 330
<210> 38
<211> 329
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 38
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 Ala 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 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
245 250 255
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
260 265 270
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
275 280 285
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
290 295 300
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
305 310 315 320
Lys Ser Leu Ser Leu Ser Pro Gly Lys
325
<210> 39
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa is Leu or any other amino acid
<400> 39
Gly Phe Thr Phe Ser Ser Tyr Xaa Met Ser
1 5 10
<210> 40
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa is Val or a conservative substitution of Val
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is Thr or Asn
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa is Ala or Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa is Glu or Asn
<220>
<221> MISC_FEATURE
<222> (11)..(11)
<223> Xaa is Thr or Lys
<220>
<221> MISC_FEATURE
<222> (14)..(14)
<223> Xaa is Pro or Val
<400> 40
Xaa Ala Xaa Ile Ser Gly Gly Gly Xaa Xaa Xaa Tyr Tyr Xaa Asp Ser
1 5 10 15
Val Lys Gly
<210> 41
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVH1
<400> 41
Glu Val Gln Leu Leu Glu 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 Ser Val Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Lys Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 42
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVH2
<400> 42
Glu Val Gln Leu Leu Glu 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 Ser Val Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Lys Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 43
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVH3
<400> 43
Glu Val Gln Leu Leu Glu 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 Ser Val Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp His Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 44
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVH4
<400> 44
Glu Val Gln Leu Leu Glu 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 Ser Val Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp His Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 45
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVH5
<400> 45
Glu Val Gln Leu Leu Glu 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 Ser Val Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp His Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 46
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVL1
<400> 46
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 Gln Ala Ser Gln Ser Ile Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Asn Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Asn Tyr Pro Leu Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 47
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVL2
<400> 47
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 Gln Ala Ser Gln Asp Ile Ser Tyr Leu
20 25 30
Tyr Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Ala Ser Asn Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Asn Tyr Pro Leu Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 48
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> MAB01-MAB15 IgG1 designer variable Domain sequence DVL3
<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 Gln Ala Ser Gln Asp Ile Ser Tyr Leu
20 25 30
Tyr Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45
Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Asn Tyr Pro Leu Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 49
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VH sequence F11VH-1
<400> 49
Glu Val Gln Leu Leu Glu 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 Ser Val Thr Ser Tyr
20 25 30
Gly Val His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Ala Ile Trp Ser Gly Gly Ser Thr Gln Tyr Asn Ala Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Ala Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 50
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VH sequence F11VH-13
<400> 50
Glu Val Gln Leu Leu Glu 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 Ser Val Thr Ser Tyr
20 25 30
Gly Val His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Ala Ile Trp Ser Gly Gly Ser Thr Gln Tyr Asn Ala Ala Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 51
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VH sequence F11VH-23
<400> 51
Glu Val Gln Leu Leu Glu 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 Val Thr Ser Tyr
20 25 30
Ala Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Phe
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 52
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VH sequence F11VH-12
<400> 52
Glu Val Gln Leu Leu Glu 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 Ser Val Thr Ser Tyr
20 25 30
Gly Val His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Phe
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Ala Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 53
<211> 119
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VH sequence F11VH-123
<400> 53
Glu Val Gln Leu Leu Glu 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 Ser Val Thr Ser Tyr
20 25 30
Gly Val His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Phe
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 54
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VK sequence F11VK-12
<400> 54
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 Gln Ala Ser Ser Ser Val Ser Phe Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Thr Tyr Pro Leu Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 55
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 06F11-V optimized variable Domain VK sequence F11VK-123
<400> 55
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 Gln Ala Ser Ser Ser Val Ser Phe Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Asp Thr Tyr Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 56
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 56
Ser Tyr Ala Met His
1 5
<210> 57
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR1
<400> 57
Ser Tyr Gly Met His
1 5
<210> 58
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 58
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ala Ser Val Lys Gly
1 5 10 15
<210> 59
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 59
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys Gly
1 5 10 15
<210> 60
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 60
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 61
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 61
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys Gly
1 5 10 15
<210> 62
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 62
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 63
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 63
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ala Ala Val Lys Gly
1 5 10 15
<210> 64
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 64
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ala Thr Val Lys Gly
1 5 10 15
<210> 65
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 65
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 66
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 66
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Val Lys Gly
1 5 10 15
<210> 67
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 67
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ser Val Lys Gly
1 5 10 15
<210> 68
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 68
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 69
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 69
Ala Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ser Val Lys Gly
1 5 10 15
<210> 70
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 70
Ala Ile Trp Ser Gly Gly Ser Thr Gln Tyr Asn Ala Ala Val Lys Gly
1 5 10 15
<210> 71
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 71
Ala Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly
1 5 10 15
<210> 72
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 72
Ala Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Asn Ala Ala Val Lys Gly
1 5 10 15
<210> 73
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 73
Ala Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Asn Ala Ser Val Lys Gly
1 5 10 15
<210> 74
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 74
Ala Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 75
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 75
Ala Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Ala Ala Ala Val Lys Gly
1 5 10 15
<210> 76
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 76
Ala Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ala Val Lys Gly
1 5 10 15
<210> 77
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 77
Ala Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Val Lys Gly
1 5 10 15
<210> 78
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 78
Ala Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 79
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 79
Ala Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Val Lys Gly
1 5 10 15
<210> 80
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 80
Ala Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ala Val Lys Gly
1 5 10 15
<210> 81
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 81
Ala Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 82
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 82
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ala Ala Val Lys Gly
1 5 10 15
<210> 83
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 83
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ala Ser Val Lys Gly
1 5 10 15
<210> 84
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 84
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Val Lys Gly
1 5 10 15
<210> 85
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 85
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ser Val Lys Gly
1 5 10 15
<210> 86
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 86
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ser Val Lys Gly
1 5 10 15
<210> 87
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 87
Val Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 88
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 88
Val Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Ala Ala Ser Val Lys Gly
1 5 10 15
<210> 89
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 89
Val Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 90
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 90
Val Ile Tyr Ser Gly Gly Ser Thr Asp Tyr Asn Asp Ala Val Lys Gly
1 5 10 15
<210> 91
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR2
<400> 91
Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Asn Ala Ala Val Lys Gly
1 5 10 15
<210> 92
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 92
Ala Gly Asp Tyr Glu Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 93
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 93
Ala Gly Asp Lys Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 94
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 94
Ala Gly Asp Gln Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 95
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 95
Ala Gly Asp Glu Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 96
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 96
Ala Gly Asp His Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 97
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 97
Ala Gly Asp Met Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 98
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 98
Ala Gly Asp Asn Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 99
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 99
Ala Gly Asp Asn Asn Tyr Asp Gly Phe Glu Tyr
1 5 10
<210> 100
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 100
Ala Gly Asp Tyr Asp Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 101
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 101
Ala Gly Asp Tyr Asn Leu Asp Gly Phe Ala Tyr
1 5 10
<210> 102
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 102
Ala Gly Asp Tyr Asn Trp Asp Gly Phe Ala Tyr
1 5 10
<210> 103
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 103
Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Ile
1 5 10
<210> 104
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 104
Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Met
1 5 10
<210> 105
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 105
Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Asn
1 5 10
<210> 106
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 106
Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Ala Trp
1 5 10
<210> 107
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 107
Ala Gly Asp Tyr Asn Tyr Asp Gly Phe Arg Tyr
1 5 10
<210> 108
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 108
Ala Gly Asp Tyr Asn Tyr Asp Gly Leu Ala Tyr
1 5 10
<210> 109
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 109
Ala Gly Asn Tyr Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 110
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 110
Ala Gly Pro Tyr Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 111
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<400> 111
Ala Gly Thr Tyr Asn Tyr Asp Gly Phe Ala Tyr
1 5 10
<210> 112
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 112
Gln Ala Ser Gln Asp Ile Ser Phe Met Tyr
1 5 10
<210> 113
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 113
Gln Ala Ser Gln Ser Val Ser His Leu Tyr
1 5 10
<210> 114
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 114
Gln Ala Ser Gln Ser Val Ser Tyr Met Tyr
1 5 10
<210> 115
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 115
Gln Ala Ser Ser Ser Ile Ser Phe Met Tyr
1 5 10
<210> 116
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 116
Gln Ala Ser Ser Ser Ile Ser Tyr Met Tyr
1 5 10
<210> 117
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 117
Gln Ala Ser Ser Ser Val Ser His Met Tyr
1 5 10
<210> 118
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 118
Gln Ala Ser Gln Ser Ile Ser Phe Leu Tyr
1 5 10
<210> 119
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 119
Gln Ala Ser Gln Ser Ile Ser His Leu Tyr
1 5 10
<210> 120
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 120
Gln Ala Ser Gln Ser Ile Ser His Met Tyr
1 5 10
<210> 121
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 121
Gln Ala Ser Ser Asp Ile Ser His Leu Tyr
1 5 10
<210> 122
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 122
Gln Ala Ser Ser Ser Val Ser His Leu Tyr
1 5 10
<210> 123
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 123
Gln Ala Ser Gln Ser Ile Ser Tyr Met Tyr
1 5 10
<210> 124
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 124
Gln Ala Ser Gln Asp Ile Ser His Leu Tyr
1 5 10
<210> 125
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 125
Gln Ala Ser Gln Asp Ile Ser Tyr Leu Tyr
1 5 10
<210> 126
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 126
Gln Ala Ser Gln Asp Ile Ser Tyr Met Tyr
1 5 10
<210> 127
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 127
Gln Ala Ser Gln Asp Val Ser Phe Leu Tyr
1 5 10
<210> 128
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 128
Gln Ala Ser Gln Asp Val Ser His Leu Asn
1 5 10
<210> 129
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 129
Gln Ala Ser Gln Asp Val Ser His Leu Tyr
1 5 10
<210> 130
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 130
Gln Ala Ser Gln Asp Val Ser His Met Tyr
1 5 10
<210> 131
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 131
Gln Ala Ser Gln Asp Val Ser Tyr Leu Tyr
1 5 10
<210> 132
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 132
Gln Ala Ser Gln Asp Val Ser Tyr Met Tyr
1 5 10
<210> 133
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 133
Gln Ala Ser Gln Ser Ile Ser Phe Met Tyr
1 5 10
<210> 134
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 134
Gln Ala Ser Gln Ser Val Arg His Met Tyr
1 5 10
<210> 135
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 135
Gln Ala Ser Gln Ser Val Ser Phe Met Tyr
1 5 10
<210> 136
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 136
Gln Ala Ser Gln Ser Val Ser His Met Tyr
1 5 10
<210> 137
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 137
Gln Ala Ser Gln Ser Val Ser Tyr Leu Tyr
1 5 10
<210> 138
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 138
Gln Ala Ser Ser Asp Ile Ser Phe Met Tyr
1 5 10
<210> 139
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 139
Gln Ala Ser Ser Asp Ile Ser Tyr Met Tyr
1 5 10
<210> 140
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 140
Gln Ala Ser Ser Asp Val Ser Phe Leu Tyr
1 5 10
<210> 141
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 141
Gln Ala Ser Ser Asp Val Ser Phe Met Tyr
1 5 10
<210> 142
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 142
Gln Ala Ser Ser Asp Val Ser His Leu Tyr
1 5 10
<210> 143
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 143
Gln Ala Ser Ser Asp Val Ser His Met Tyr
1 5 10
<210> 144
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 144
Gln Ala Ser Ser Asp Val Ser Tyr Met Tyr
1 5 10
<210> 145
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 145
Gln Ala Ser Ser Ser Ile Ser Phe Leu Tyr
1 5 10
<210> 146
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 146
Gln Ala Ser Ser Ser Ile Ser His Leu Tyr
1 5 10
<210> 147
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 147
Gln Ala Ser Ser Ser Ile Ser Tyr Leu Tyr
1 5 10
<210> 148
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<400> 148
Gln Ala Ser Ser Ser Val Ser Tyr Met Tyr
1 5 10
<210> 149
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 149
Asp Ala Ser Asn Leu Ala Thr
1 5
<210> 150
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 150
Asp Thr Ser Asn Leu Ala Thr
1 5
<210> 151
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 151
Asp Thr Ser Asn Leu Glu Thr
1 5
<210> 152
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<400> 152
Asp Ala Ser Asn Leu Glu Thr
1 5
<210> 153
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 153
Gln Gln Trp Asp Asn Tyr Pro Leu Thr
1 5
<210> 154
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 154
Gln Gln Trp Asp Thr Tyr Pro Leu Thr
1 5
<210> 155
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 155
Gln Gln Trp Ser Asn Tyr Pro Leu Thr
1 5
<210> 156
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 156
Gln Gln Trp Asp Asn Leu Pro Leu Thr
1 5
<210> 157
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 157
Gln Gln Trp Asp Thr Leu Pro Leu Thr
1 5
<210> 158
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 158
Gln Gln Trp Ser Asn Leu Pro Leu Thr
1 5
<210> 159
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<400> 159
Gln Gln Trp Ser Thr Leu Pro Leu Thr
1 5
<210> 160
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> HCDR3
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa is Gln or any other amino acid
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa is Leu or any other amino acid
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is Tyr or a conservative substitution of Tyr
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa is Tyr or any other amino acid
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa is Phe or any other amino acid
<400> 160
Xaa Xaa Xaa Xaa Xaa Asp Tyr
1 5
<210> 161
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR1
<220>
<221> MISC_FEATURE
<222> (7)..(8)
<223> Xaa is Ser or a conservative substitution of Ser
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa is Tyr or a conservative substitution of Tyr
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa is Leu or a conservative substitution of Leu
<220>
<221> MISC_FEATURE
<222> (11)..(11)
<223> Xaa is Asn or Thr or a conservative substitution of Asn or Thr
<400> 161
Arg Ala Ser Gln Ser Ile Xaa Xaa Xaa Xaa Xaa
1 5 10
<210> 162
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR2
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa is Ala or Thr or a conservative substitution of Ala or Thr
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is Ser or a conservative substitution of Ser
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa is Gln or any other amino acid
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> Xaa is Ser or any other amino acid
<400> 162
Xaa Ala Xaa Ser Leu Xaa Xaa
1 5
<210> 163
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> LCDR3
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is Ser or any other amino acid
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa is Thr or any other amino acid
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa is Trp or any other amino acid
<400> 163
Gln Gln Xaa Tyr Ser Xaa Pro Xaa Thr
1 5

Claims (31)

1. An anti-CD 122 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
(a) The VH region amino acid sequence comprises HCDR1 comprising SEQ ID NO. 3, HCDR2 comprising SEQ ID NO. 5 and HCDR3 comprising SEQ ID NO. 7 and the VL region amino acid sequence comprises LCDR1 comprising SEQ ID NO. 18, LCDR2 comprising SEQ ID NO. 13 and LCDR3 comprising SEQ ID NO. 15; or alternatively
(b) The amino acid sequence of the VH region comprises HCDR1 containing SEQ ID NO. 3, HCDR2 containing SEQ ID NO. 5 and HCDR3 containing SEQ ID NO. 7; and the VL amino acid sequence comprises LCDR1 comprising SEQ ID NO. 11, LCDR2 comprising SEQ ID NO. 13 and LCDR3 comprising SEQ ID NO. 15.
2. The antibody or antigen binding portion of claim 1, wherein
(a) The VH region amino acid sequence comprises SEQ ID NO. 1 and the VL region amino acid sequence comprises SEQ ID NO. 17; or alternatively
(b) The VH region amino acid sequence comprises SEQ ID NO. 1 and the VL region amino acid sequence comprises SEQ ID NO. 9.
3. The antibody or antigen-binding portion of claim 1 or 2, wherein the antibody or antigen-binding portion is humanized or chimeric.
4. The antibody or antigen-binding portion of any one of claims 1-3, wherein the VH region, the VL region, or both the VH region and the VL region comprise one or more human framework region amino acid sequences.
5. The antibody or antigen-binding portion of any one of claims 1-4, wherein the VH region, the VL region, or both the VH region and the VL region comprise a human variable region framework scaffold amino acid sequence into which the CDR amino acid sequence has been inserted.
6. The antibody or antigen-binding portion of any one of claims 1 and 3-5, wherein the VH region comprises an IGHV3-23 human germline scaffold amino acid sequence into which the HCDR1, HCDR2 and HCDR3 amino acid sequences have been inserted.
7. The antibody or antigen binding portion of any one of claims 1 and 3-6, wherein the VL region comprises an IGKV1-33 human germline scaffold amino acid sequence into which the LCDR1, LCDR2, and LCDR3 amino acid sequences have been inserted.
8. The antibody or antigen-binding portion of any one of claims 1-7, wherein the antibody comprises an immunoglobulin constant region.
9. The antibody or antigen binding portion of claim 8, wherein the immunoglobulin constant region is IgG, igE, igM, igD, igA or IgY.
10. The antibody or antigen binding portion of claim 9, wherein the immunoglobulin constant region is IgG1, igG2, igG3, igG4, igA1, or IgA2.
11. The antibody or antigen binding portion of claim 8, wherein the immunoglobulin constant region is immunologically inert.
12. The antibody or antigen-binding portion of claim 8, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising amino acid substitutions S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A, L235A and G237A, or a wild-type human IgG2 constant region, wherein numbering is according to the EU index as in Kabat.
13. The antibody or antigen-binding portion of claim 8, wherein the immunoglobulin constant region comprises any of SEQ ID NOs 32-38.
14. The antibody or antigen-binding portion of any one of claims 1-13, wherein the antibody or antigen-binding portion is Fab, fab ', F (ab') 2 Fv, scFv, macroantibody, minibody, diabody, triabody, tetrabody or diabody.
15. The antibody or antigen binding portion of any one of claims 1-14, wherein the antibody is monoclonal.
16. The antibody or antigen-binding portion of any one of claims 1-15, wherein the antibody is a tetrameric antibody, a tetravalent antibody, or a multispecific antibody.
17. The antibody or antigen-binding portion of any one of claims 1-16, wherein the antibody is a bispecific antibody that specifically binds a first antigen and a second antigen, wherein the first antigen is CD122 and the second antigen is not CD122.
18. An immunoconjugate comprising the antibody or antigen binding portion of any one of claims 1-17 linked to a therapeutic agent.
19. The immunoconjugate of claim 18, wherein the therapeutic agent is a cytotoxin, radioisotope, chemotherapeutic agent, immunomodulatory agent, cytostatic enzyme, cytolytic enzyme, therapeutic nucleic acid, anti-angiogenic agent, anti-proliferative agent, or pro-apoptotic agent.
20. A pharmaceutical composition comprising the antibody or antigen-binding portion of any one of claims 1-17 or the immunoconjugate of claim 18 or 19, and a pharmaceutically acceptable carrier, diluent, or excipient.
21. A nucleic acid molecule encoding the antibody or antigen-binding portion of any one of claims 1-17
(a) VH region amino acid sequence;
(b) VL region amino acid sequence; or (b)
(c) VH region amino acid sequence and VL region amino acid sequence.
22. An expression vector comprising the nucleic acid molecule of claim 21.
23. A recombinant host cell comprising the nucleic acid molecule of claim 21 or the expression vector of claim 22.
24. A method of producing an anti-CD 122 antibody, or antigen-binding portion thereof, comprising:
culturing a recombinant host cell comprising the expression vector of claim 22 under conditions that express the nucleic acid molecule, thereby producing the antibody or antigen-binding portion; and
isolating the antibody or antigen binding portion from the host cell or culture.
25. A method for inhibiting an immune response in a subject comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding portion of any one of claims 1-17, the immunoconjugate of claim 18 or 19, or the pharmaceutical composition of claim 20.
26. The method of claim 25, wherein the immune response is mediated by CD 122.
27. A method for treating or preventing a disease in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding portion of any one of claims 1-17, the immunoconjugate of claim 18 or 19, or the pharmaceutical composition of claim 20.
28. The method of claim 27, wherein the disease is an inflammatory disease or an autoimmune disease.
29. The method of claim 27, wherein the disease is vitiligo, celiac disease, type 1 diabetes, multiple sclerosis, graft versus host disease, systemic lupus erythematosus, psoriasis, atopic dermatitis, alopecia areata, ulcerative colitis, or rheumatoid arthritis.
30. A method for inhibiting IL-15-induced T cell migration from skin, the method comprising contacting the skin with a therapeutically effective amount of the antibody or antigen-binding portion of any one of claims 1-17, the immunoconjugate of claim 18 or 19, or the pharmaceutical composition of claim 20.
31. The antibody or antigen-binding portion of any one of claims 1-17, the immunoconjugate of claim 18 or 19, or the pharmaceutical composition of claim 20 for use as a medicament.
CN202280028157.8A 2021-04-14 2022-04-13 anti-CD 122 antibodies and uses thereof Pending CN117295762A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63/174,772 2021-04-14
US202163279762P 2021-11-16 2021-11-16
US63/279,762 2021-11-16
PCT/US2022/024620 WO2022221409A1 (en) 2021-04-14 2022-04-13 Anti-cd122 antibodies and uses thereof

Publications (1)

Publication Number Publication Date
CN117295762A true CN117295762A (en) 2023-12-26

Family

ID=89239467

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN117295762A (en)

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