TW201326212A - Anti-MCSP antibodies - Google Patents

Abstract

The invention provides anti-MCSP antibodies and methods of using the same.

Description

anti-MCSP antibody

The present invention relates to anti-MCSP antibodies and methods of using the same to treat and diagnose diseases.

The present application claims priority to European Patent Application No. EP 11178393.2, filed on Aug. 23, 2011, the disclosure of which is hereby incorporated by reference.

MCSP

Melanoma chondroitin sulfate proteoglycan (MCSP) is a large transmembrane proteoglycan that is expressed in most melanoma cancers. MCSP is also manifested in other cancers, including glioma blastoma, osteosarcoma, chondrosarcoma, some types of ALL and AML, and basal cell carcinoma. It is used as an early cell surface melanoma progression marker and is involved in stimulating tumor cell proliferation, metastasis, migration, invasion, and angiogenesis. Staube, E., et al, FEBS Letters, 527: 114-118 (2002); Campoli, M. et al, Crit. Rev. Immun. 24: 267-296 (2004); Vergilis, IJ, J Invest Dermatol, 125 : 526-531 (2005); Yang, J., JCB, 165: 881-891 (2004); Luo, W., J. Immuno., 176: 6046-6054 (2006).

Antibody glycosylation

Oligosaccharide components can significantly affect properties associated with the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activities. These properties may depend not only on the presence or absence of oligosaccharides, but also on the specificity of the oligosaccharides. structure. Some generalizations can be made between oligosaccharide structure and glycoprotein function. For example, certain oligosaccharide structures mediate rapid clearance of glycoproteins from the bloodstream via interaction with specific carbohydrate binding proteins, while other oligosaccharide structures can bind to antibodies and trigger undesired immune responses (Jenkins et al., Nat Biotechnol 14, 975-81 (1996)).

An IgGl type antibody (the most commonly used antibody in cancer immunotherapy) is a glycoprotein having a conserved N-linked glycosylation site at Asn 297 of each CH2 domain. Two complex biantennary oligosaccharides attached to Asn 297 are hidden between the CH2 domains, forming extensive contact with the polypeptide backbone, and their presence is mediated by antibody-dependent effector functions (eg, antibody-dependent cell-mediated cytotoxicity) (ADCC)) is critical (Lifely et al, Glycobiology 5, 813-822 (1995); Jefferis et al, Immunol Rev 163, 59-76 (1998); Wright and Morrison, Trends Biotechnol 15, 26-32 (1997)) .

The cell-mediated effector function of a monoclonal antibody can be enhanced by engineering its oligosaccharide component, as described in Umana et al., Nat Biotechnol 17, 176-180 (1999) and U.S. Patent No. 6,602,684 (WO 99/54342). . Umana et al. showed that β(1,4)-N-acetylglucosamine transferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, is present in Chinese hamster ovary (CHO) cells. Overexpression significantly increased the in vitro ADCC activity of antibodies produced in these cells. Alteration of Asn 297 carbohydrates in the composition or its elimination also affects the binding of the antibody Fc-domain to Fc.γ.R and C1q proteins (Umana et al., Nat Biotech) Ol 17, 176-180 (1999); Davies et al, Biotechnol Bioeng 74, 288-294 (2001); Mimura et al, J Biol Chem 276, 45539-45547 (2001); Radaev et al, J Biol Chem 276, 16478-16483 ( 2001); Shields et al, J Biol Chem 276, 6591-6604 (2001); Shields et al, J Biol Chem 277, 26733-26740 (2002); Simmons et al, J Immunol Methods 263, 133-147 (2002)).

The invention provides anti-MCSP antibodies and methods of use thereof. One aspect of the invention provides an isolated antibody that binds to a proximal epitope of a human MCSP, wherein the antibody has been glycosylated to modify an oligosaccharide in the Fc region, and wherein the antibody has and has not been glycosylated The antibody is compared to the enhanced ADCC effector function. In one embodiment, the membrane proximal epitope of human MCSP comprises a domain comprising a CSPG repeat. In one embodiment, the domain comprising a CSPG repeat comprises a CSPG repeat 14 (SEQ ID NO: 3). In one embodiment, the Fc region of the antibody has a reduced number of fucose residues compared to an antibody that has not been glycosylated. In one embodiment, the Fc region of the antibody has an increased ratio of GlcNAc residues to fucose residues as compared to an antibody that has not been glycosylated. In one embodiment, the Fc region of the antibody has a ratio of dichomeric oligosaccharides that are increased compared to antibodies that have not been glycosylated. In certain embodiments, the anti-system monoclonal antibody. In certain embodiments, the anti-systematic human, humanized or chimeric antibody. In certain embodiments, the anti-systemic full length IgG class antibody.

In one embodiment, the anti-MCSP antibody comprises HVR-H1 comprising the amino acid sequence SEQ ID NO: 14, HVR-H2 comprising the amino acid sequence SEQ ID NO: 15 and comprising the amino acid sequence SEQ ID NO: 16 of the HVR-H3. In one embodiment, the anti-MCSP antibody comprises HVR-L1 comprising an amino acid SEQ ID NO: 10; HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and HVR-L3 comprising the amino acid sequence SEQ ID NO: 12. In one embodiment, the anti-MCSP antibody comprises HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; HVR-H2 comprising the amino acid sequence SEQ ID NO: 15; HVR-H3 comprising an amine a base acid sequence of SEQ ID NO: 16; HVR-L1 comprising an amino acid sequence of SEQ ID NO: 10; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 11; and HVR-L3 comprising an amine group Acid sequence SEQ ID NO:12.

In one embodiment, the anti-MCSP antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and comprising the amino acid sequence SEQ ID NO: 16 of the HVR-H3. In one embodiment, the anti-MCSP antibody comprises HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and HVR-L3, comprising Amino acid sequence SEQ ID NO: 12. In one embodiment, the anti-MCSP antibody comprises HVR-H1 comprising the amino acid sequence SEQ ID NO: 17; HVR-H2 comprising the amino acid sequence SEQ ID NO: 18; HVR-H3 comprising an amine a base acid sequence of SEQ ID NO: 16; HVR-L1 comprising an amino acid sequence of SEQ ID NO: 13; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 11; and HVR-L3 comprising an amine group Acid sequence SEQ ID NO:12.

In one embodiment, the anti-MCSP antibody comprises a VH sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:29; and at least 95% sequence identity to the amino acid sequence SEQ ID NO:28 a VL sequence; or a VH having at least 95% sequence identity to the amino acid sequence SEQ ID NO:29 The sequence and the VL sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:28.

In one embodiment, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO:29; the VL sequence of SEQ ID NO:28. In one embodiment, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO:29 and the VL sequence of SEQ ID NO:28.

In one embodiment, the anti-MCSP antibody comprises a VH sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 32; and at least 95% sequence identity to the amino acid sequence SEQ ID NO: 31 a VL sequence; or a VH sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 32 and a VL sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:32.

In one embodiment, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO:29; the VL sequence of SEQ ID NO:28. In one embodiment, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO:29 and the VL sequence of SEQ ID NO:28.

Another aspect of the invention provides an isolated nucleic acid encoding the above anti-MCSP antibody. Another aspect of the invention provides a host cell comprising the nucleic acid. Another aspect of the invention provides a method of producing an antibody comprising culturing the host cell to produce the antibody.

Another aspect of the invention provides an immunoconjugate comprising the anti-MCSP antibody described above and a cytotoxic agent. Another aspect of the invention provides an immunoconjugate comprising the anti-MCSP antibody described above and a pharmaceutically acceptable carrier.

Another aspect of the invention provides an immunoconjugate comprising the above anti-MCSP antibody A combination that is used as a medicament. Another aspect of the present invention provides the above anti-MCSP antibody or immunoconjugate thereof for use in the treatment of cancer, particularly cancers which exhibit MCSP, including skin cancer (including melanoma and basal cell carcinoma), glial Tumors (including glioma blastoma), bone cancers (such as osteosarcoma), and leukemia (including ALL and AML).

Another aspect of the invention provides the use of the above anti-MCSP antibody for inducing cell lysis. Another aspect of the invention provides the use of the above anti-MCSP antibody or immunoconjugate thereof for the manufacture of a medicament (e.g., an agent for treating cancer) or for inducing cell lysis.

Another aspect of the invention provides a method of treating an individual having cancer comprising administering to the individual an effective amount of the above anti-MCSP antibody or immunoconjugate thereof. Cancer systems, for example, cancers that exhibit MCSP, such as skin cancer (including melanoma and basal cell carcinoma), glioma (including neutrophil blastoma), bone cancer (such as osteosarcoma), and leukemia (including ALL and AML) ).

Another aspect of the invention provides a method of inducing cell lysis in an individual comprising administering to the individual an effective amount of the above anti-MCSP antibody or immunoconjugate thereof to induce cell lysis.

I. Definition

For the purposes herein, "receptor human framework" is an amino acid comprising a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. The framework of the sequence, as defined below. The acceptor human framework "derived from" the human immunoglobulin framework or the human consensus framework may comprise the same amino acid sequence, or it may contain an amino acid Sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or Less or 2 or less. In some embodiments, the sequence of the VL receptor human framework is identical to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, an antibody and an antigen). The affinity of the molecule X for its partner Y can usually be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are set forth below.

An "affinity mature" anti-system refers to an antibody having such alterations as compared to a parent antibody that does not have one or more alterations in one or more hypervariable regions (HVR), which alters the affinity of the antibody for the antigen. Sex.

"Angiogenic disorder" means any vascular dysfunction, including non-neoplastic and neoplastic conditions. Tumorous conditions include, but are not limited to, those described below. Non-tumorous conditions include, but are not limited to, undesired or abnormal hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, plaque psoriasis, sarcoma-like, atherosclerosis, atherosclerotic plaque , diabetes and other proliferative retinopathy (including retinopathy of prematurity, post-lens fibrosis, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft regeneration Vascular, corneal graft rejection, retinal/choroidal neovascularization, neovascularization of the angle, red blood vessel disease, vascular restenosis, arteriovenous malformation (AVM), meningioma, blood vessels Tumor, angiofibroma, thyroid hyperplasia (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, pneumonia, acute lung injury/ARDS, sepsis, primary pulmonary hypertension, malignant lung Fluid, cerebral edema (for example, associated with acute stroke/closed head injury/trauma), bursitis inflammation, vasospasm formation in RA, ossifying myositis, hypertrophic bone formation, osteoarthritis (OA) , refractory ascites, polycystic ovarian disease, endometriosis, third gap of fluid imbalance (pancreatitis, chamber syndrome, burns, bowel disease), uterine fibroids, premature delivery, chronic inflammation (eg IBD (clone Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, renal disease, undesired or abnormal tissue mass growth (non-cancer), hemorrhagic joints, hypertrophy Scar, hair growth inhibition, Osler-Weber syndrome, suppurative granuloma, posterior lens fibrosis, scleroderma, trachoma, vascular adhesion, synovitis, dermatitis, pre-eclampsia, ascites , pericardial effusion (for example, associated with pericarditis) and pleural effusion.

The terms "anti-MCSP antibody" and "antibody that binds to MCSP" refer to an antibody that binds MCSP with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting MCSP. In one embodiment, the anti-MCSP antibody binds to an unrelated non-MCSP protein to a lesser extent than about 10% of the antibody binds to MCSP, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that binds to MCSP 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM (eg 10 ^-8 M or lower, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M). In certain embodiments, the anti-MCSP antibody binds to an epitope in MCSP that is conserved between MCSPs from different species.

The term "antibody" is used herein in its broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as It can exhibit the desired antigen binding activity.

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

An antibody that "binds to the same epitope as a reference antibody" refers to an antibody that blocks 50% or more of the binding of a reference antibody to its antigen in a competition assay, and conversely, the reference antibody binds the antibody to its antigen in a competition assay. Block 50% or more. An example competition analysis is provided herein.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by a disorder of cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (eg, Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, bone cancer (eg osteosarcoma, chondrosarcoma, Yiwen) Sarcoma (Ewing's Sarcoma)), gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney Cancer, liver cancer, prostate cancer, skin cancer (such as melanoma and basal cell carcinoma), vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders and various types of head and neck cancer.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a certain degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

The term "chimeric" anti-system refers to a portion of a heavy chain and/or a light chain that is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from antibodies of different sources or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five major categories of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy-chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ^{212 ,} and Lu); Chemotherapeutic agents or drugs (eg, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan (melphalan), mitomycin C, chlorambucil, daunorubicin or other intercalating agents; growth inhibitors; enzymes and fragments thereof, such as lysing enzymes; Antibiotics; toxins, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed below.

"Effector function" refers to the biological activity attributed to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) Bottom); and B cell activation.

An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result at the desired dosage and for the desired period of time.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminus of the Fc region may or may not be present in the amine acid (Lys447). Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system (also known as the EU index), as in Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition, Public. Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to a variable domain residue other than a hypervariable region (HVR) residue. The FR of the variable domain is typically composed of four FR domains FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences are typically found in the following sequences in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny and the parental cell may not be identical and may contain mutations. Included herein are mutant progeny that have been screened or selected for the same function or biological activity as the original transformed cell.

A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or an antibody obtained from a non-human source using a human antibody profile or other human antibody coding sequence. The definition of this human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

The "Human Common Framework" represents the framework of the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the human immunoglobulin VL or VH sequence is selected from a subset of variable domain sequences. Typically, subgroups of sequences are as set forth in Kabat et al, Sequences of Proteins of Immunological Interest , 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is as described above for the subgroup kappa of Kabat et al. In one embodiment, for VH, the subgroup is as described above for subgroup III of Kabat et al.

A "humanized" anti-system refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the HVRs (eg, CDRs) correspond to non-humans, and all or Essentially all FRs correspond to those of human antibodies. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The term "hypervariable region" or "HVR" as used herein, refers to each of the regions of the antibody variable domain region that are highly variable in sequence and/or form a structurally defined loop ("hypervariable loop"). Typically, the native four-chain antibody comprises six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) which have the highest sequence variability and/or participate in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101. (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplified CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) Amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al, Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).) In addition to CDR1 in VH, CDRs generally comprise an amine group that forms a hypervariable loop. Acid residue. The CDR also contains a "specificity determining residue" or "SDR" which is a residue that contacts the antigen. The SDR is contained within the region of the CDR called the abbreviated-CDR or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur in the amino acid residue of L1 Base 31-34, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2 and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues (eg, FR residues) in the variable domain are described herein. Numbered according to the above Kabat et al.

An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

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

The "separated" anti-system has been separated from its natural environment components. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electroporation Swim) or chromatographic (eg, ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al, J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its natural environment. An isolated nucleic acid includes the nucleic acid molecule contained in a cell that typically contains a nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-MCSP antibody" refers to one or more nucleic acid molecules encoding an antibody heavy and light chain (or a fragment thereof), including the nucleic acid molecule in a single vector or in a plurality of separate vectors, The nucleic acid molecule is present at one or more locations in the host cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies, eg, containing natural mutations Or variants produced during the production of a monoclonal antibody preparation, such variants are typically present in minor amounts. Each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen as compared to a multi-drug antibody preparation that typically includes different antibodies directed against different determinants (epitopes). Thus, the modifier "single plant" indicates that the antibody is characterized by a population of antibodies that are substantially homologous and should not be considered to require production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques including, but not limited to, fusion knob methods, recombinant DNA methods, phage display methods, and utilization of all or part of a human immunoglobulin locus. Methods of transgenic animals, such methods and other exemplary methods of preparing monoclonal antibodies are set forth herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radioactive label. Naked antibodies can be stored in pharmaceutical formulations.

"Native antibody" refers to a natural immunoglobulin molecule having a different structure. For example, a native IgG anti-system heterologous tetrameric glycoprotein of about 150,000 daltons consists of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain in turn has a variable region (VH, also known as a variable heavy chain domain or a heavy chain variable domain) and three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain in turn has a variable region (VL, also referred to as a variable light chain domain or a light chain variable domain) and a constant light chain (CL) domain. Based on the amino acid sequence of the constant domain of the antibody, the light chain of the antibody can be assigned to one of two types, referred to as kappa type (κ) and lambda type (λ).

The term "package insert" as used herein refers to instructions that are typically included in commercial packages of therapeutic products, which contain warnings about indications, usage, dosage, administration, combination therapies, contraindications, and/or use of such therapeutic products. News.

The "percent amino acid sequence identity (%)" relative to the reference polypeptide sequence is defined as the amine in the candidate sequence and the reference polypeptide sequence after aligning the sequence and introducing a spacer (if necessary) to achieve a maximum percent sequence identity. The percentage of amino acid residues that are identical to the acid residue, and any conservative substitutions are not considered part of the sequence identity. For the purpose of determining the percent identity of the amino acid sequence, the alignment can be accomplished in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). )software. They are familiar Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the value of % amino acid sequence identity is obtained using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was designed by Genentech and the source code has been filed with user files in U.S. Copyright Office Washington D.C., 20559, which is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program should be compiled for UNIX operating systems (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are unchanged.

In the case where ALIGN-2 is used to compare an amino acid sequence, the amino acid sequence identity % of a given amino acid sequence A relative to (to, with or again) a given amino acid sequence B (or it can be expressed) A given amino acid sequence A) having or containing % identity of a certain amino acid sequence relative to a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y where X is by sequence ratio The program ALIGN-2 is rated as the number of identically matched amino acid residues in the alignment of A and B in the program, and wherein the total number of amino acid residues in the Y system B is. It will be appreciated that if the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % identity of A with respect to the amino acid sequence of B will not be equal to the % identity of B with respect to the amino acid sequence of A. % amino acid sequence identity used herein, unless otherwise specifically stated Values are obtained using the ALIGN-2 computer program as described in the previous paragraph.

The term "pharmaceutical formulation" means a formulation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain other components that are unacceptably toxic to the individual to which the formulation is administered.

"Pharmaceutically acceptable carrier" means a component of a pharmaceutical formulation that is not toxic to the individual other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

The term "MCSP" as used herein, unless otherwise indicated, refers to any natural MCSP from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). (melanoma chondroitin sulfate proteoglycan). The term encompasses "full length" untreated MCSP and any form of MCSP produced from processing in the cell. The term also encompasses natural variants of MCSP, such as splice variants or dual gene variants. MCSP is also known as chondroitin sulfate proteoglycan 4 (CSPG4), chondroitin sulfate proteoglycan NG2, high molecular weight melanoma associated antigen (HMW-MAA), and melanoma chondroitin sulfate proteoglycan. The amino acid sequence of an exemplary human MCSP is shown in SEQ ID NO: 1. See also Pluschke G., et al, Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan, Proc. Natl. Acad. Sci. USA 93:9710-9715 (1996); Staub E. et al., A novel repeat in the Melanoma-associated chondroitin sulfate proteoglycan defines a new protein family, FEBS Lett. 527: 114-118 (2002); Genbank Accession No: NP_001888.

As used herein, "treatment" (and its grammatical variations, such as "treat" or "treating" refers to a clinical intervention that attempts to alter the natural course of the individual being treated and may be performed for prophylactic purposes or during the course of clinical pathology. Consensus effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological outcome of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or mitigating the disease state, and ameliorating or improving the prognosis. In some embodiments, antibodies of the invention are used to delay the onset of disease or slow the progression of the disease.

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

The term "vector," as used herein, refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are included in the genome of the host cell into which the vector has been introduced. Certain vectors are capable of directing the performance of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression carriers."

II. Compositions and methods

The present invention provides anti-MCSP antibodies useful for the treatment and/or diagnosis of cell proliferative diseases such as cancer. In certain embodiments, an antibody that binds to a proximal epitope of a membrane of MCSP is provided. In certain embodiments, an antibody having enhanced effector function in combination with MCSP is provided.

A. Exemplary anti-MCSP antibodies

In one aspect, the invention provides an isolated antibody that binds to MCSP. In particular, the anti-MCSP antibodies provided herein bind to the proximal epitope of the membrane of human MCSP. As discussed in Staub E., et al, FEBS Lett. 527: 114-118 (2002), the membrane proximal region of MCSP comprises a plurality of novel repeat domain (referred to as CSPG repeat domain). image 3. The anti-MCSP antibodies of the invention bind to an epitope present in the proximal domain of the membrane of human MCSP, the membrane proximal domain comprising a domain comprising a CSPG repeat. In one embodiment, the domain comprising the CSPG repeat comprises a CSPG repeat 14 corresponding to the amino acid 1937-2043 of human MCSP. In one embodiment, the CSPG repeat 14 domain has the amino acid sequence set forth in SEQ ID NO:3. In another embodiment, the domain comprising a CSPG repeat comprises at least a portion of a CSPG repeat 14 and a CSPG repeat 15. The CSPG repeat 15 domain corresponds to the amino acid 2044-2246 of human MCSP. In one embodiment, the CSPG repeat -15 domain has the amino acid sequence SEQ ID NO:4. In one embodiment, the domain comprising a CSPG repeat comprises the amino acid sequence SEQ ID NO:5. In one embodiment, the domain comprising a CSPG repeat comprises the amino acid sequence SEQ ID NO: 5 and is free of the native transmembrane domain. In one embodiment, the domain comprising the CSPG repeat comprises the CSPG repeats 13-15. In a real In the example, the domain containing the CSPG repeat comprises the amino acid sequence SEQ ID NO: 6. In one embodiment, the domain comprising a CSPG repeat comprises the amino acid sequence SEQ ID NO: 6 and is free of a native transmembrane domain. In one embodiment, the domain comprising the CSPG repeat comprises CSPG repeats 12-15. In one embodiment, the domain comprising a CSPG repeat comprises the amino acid sequence SEQ ID NO: 7. In one embodiment, the domain comprising a CSPG repeat comprises the amino acid sequence SEQ ID NO: 7 and is free of a native transmembrane domain. In certain embodiments, the native transmembrane domain is VIPMMC LVLLLLALIL PLLFY (UniProt Accession No. Q6UVK1) (SEQ ID NO: 44).

In one embodiment, the anti-MCSP antibody induces cell lysis that exhibits MCSP. Cleavage can be induced by any mechanism, for example by mediating effector functions (eg, C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytosis; cell surface receptors (eg, B cell receptors) down-regulated; and B cell activation) are induced, or induced by direct induction of cell apoptosis.

In one embodiment, the anti-MCSP antibody is glycosylated to enhance at least one effector function compared to a parent anti-MCSP antibody that has not been glycosylated. Enhancement of effector function enhances binding affinity to Fc receptor; enhanced antibody-dependent cellular cytotoxicity (ADCC); enhanced binding to NK cells; enhanced binding to macrophages; enhanced and polymorphic Binding of nuclear cells; enhanced binding to monocytes; direct signal transduction to induce apoptosis; enhanced dendritic cell maturation; or enhanced T cell priming. Glycosylated anti-MCSP antibody in cancer with MCSP Survival benefits are provided in individuals with antibodies that are not glycosylated against the same MCSP epitope.

In one aspect, the invention provides an anti-MCSP antibody comprising at least 1, 2, 3, 4, 5 or 6 HVRs selected from the group consisting of: (a) HVR-H1 comprising an amine a base acid sequence of SEQ ID NO: 14; (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 16; HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (e) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (f) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In one aspect, the invention provides an anti-MCSP antibody comprising at least one, at least two or all three VH HVRs selected from the group consisting of: (a) HVR-H1 comprising an amino acid sequence SEQ ID NO: 14; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 15; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 15; HVR-H3 comprising the amino acid sequence SEQ ID NO: 16.

In one aspect, the invention provides an anti-MCSP antibody comprising at least one, at least two or all three VL HVRs selected from the group consisting of: (a) HVR-L1 comprising an amino acid sequence SEQ ID NO: 10; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (b) HVR- L2 comprising the amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, the anti-MCSP antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVRs selected from the group consisting of: (i) HVR-H1 comprising an amine a base acid sequence of SEQ ID NO: 14, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 15, and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: And (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10, (ii) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, the invention provides an anti-MCSP antibody comprising (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2 comprising an amino acid sequence SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (e) HVR-L2, It comprises the amino acid sequence SEQ ID NO: 11; and (f) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In one aspect, the invention provides an anti-MCSP antibody comprising at least 1, 2, 3, 4, 5 or 6 HVRs selected from the group consisting of: (a) HVR-H1 comprising an amine The acid sequence SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16; (d) HVR-L1, which contains an amine The acid sequence SEQ ID NO: 13; (e) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (f) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In one aspect, the invention provides an anti-MCSP antibody comprising at least one, at least two or all three VH HVRs selected from the group consisting of: (a) HVR-H1 comprising an amino acid sequence SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 18; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16. In another embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 18; HVR-H3 comprising the amino acid sequence SEQ ID NO: 16.

In one aspect, the invention provides an anti-MCSP antibody comprising at least one, at least two or all three VL HVRs selected from the group consisting of: (a) HVR-L1 comprising an amino acid sequence SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (c) HVR-L3, which comprises the amino acid sequence SEQ ID NO: 12.

In another aspect, the anti-MCSP antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVRs selected from the group consisting of: (i) HVR-H1 comprising an amine a base acid sequence of SEQ ID NO: 17, (ii) HVR-H2 comprising an amino acid sequence of SEQ ID NO: 18, and (iii) HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: ; And (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13, (ii) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, the invention provides an anti-MCSP antibody comprising (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising an amino acid sequence SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (e) HVR-L2, It comprises the amino acid sequence SEQ ID NO: 11; and (f) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, the invention provides an anti-MCSP antibody comprising (a) HVR-H1 comprising an amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising an amino acid sequence SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (e) HVR-L2, It comprises the amino acid sequence SEQ ID NO: 11; and (f) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, the invention provides an anti-MCSP antibody comprising (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2 comprising an amino acid sequence SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (e) HVR-L2, Amino acid sequence SEQ ID NO: 11; and (f) HVR-L3, which comprises the amino acid sequence SEQ ID NO: 12.

In another aspect, the invention provides an anti-MCSP antibody comprising (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2 comprising an amino acid sequence SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16; (d) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (e) HVR-L2, It comprises the amino acid sequence SEQ ID NO: 11; and (f) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In one aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and the amino acid sequence SEQ ID NO: 99% or 100% sequence consistent VH sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, a conservative substitution), insertion or deletion, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:27. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO: 27, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2, comprising Amino acid sequence SEQ ID NO: 15; and (c) HVR-H3 comprising an amino acid sequence SEQ ID NO: 16.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, and the amino acid sequence SEQ ID NO: 97%, 98%, 99% or 100% sequence-consistent light chain variable domain (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, a conservative substitution), insertion or deletion, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO 26. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the VL sequence of SEQ ID NO: 26, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (b) HVR-L2, comprising Amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises a VH of any of the embodiments as provided above and a VL of any of the embodiments as provided above. In one embodiment, the antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 27 and a VL sequence of SEQ ID NO: 26, including post-translational modifications of the sequences.

In another aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, and the amino acid sequence SEQ ID NO: 96%, 97%, 98%, 99% or 100% sequence consistent VH sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, a conservative substitution), insertion or deletion, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:32. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO: 32, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 17; (b) HVR-H2, comprising Amino acid sequence SEQ ID NO: 18; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16.

In another aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence SEQ ID NO:31. VL sequence with 99% or 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, a conservative substitution), insertion or deletion, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:31. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Depending on the situation, anti- The MCSP antibody comprises the VL sequence of SEQ ID NO: 31, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (b) HVR-L2, comprising Amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises a VH of any of the embodiments as provided above and a VL of any of the embodiments as provided above. In one embodiment, the antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 32 and a VL comprising the amino acid sequence SEQ ID NO: 31, including post-translational modifications of the sequences.

In another aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence SEQ ID NO:29. , 99% or 100% sequence consistent VH sequence. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, a conservative substitution), insertion or deletion, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:29. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the VH sequence of SEQ ID NO: 29, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2, comprising Amino acid sequence SEQ ID NO: 18; and (c) HVR-H3, which comprises the amino acid sequence SEQ ID NO: 16.

In another aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence SEQ ID NO:28. VL sequence with 99% or 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence ( For example, a conservative substitution), insertion or deletion, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:28. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the VL sequence of SEQ ID NO: 28, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (b) HVR-L2, comprising Amino acid sequence SEQ ID NO: 11; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises a VH of any of the embodiments as provided above and a VL of any of the embodiments as provided above. In one embodiment, the antibody comprises a VH comprising the amino acid sequence SEQ ID NO: 29 and a VL comprising the amino acid sequence SEQ ID NO: 28, including post-translational modifications of the sequences.

In another aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, and the amino acid sequence SEQ ID NO: Heavy chain sequence of 96%, 97%, 98%, 99% or 100% sequence identity. In certain embodiments, a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence (eg, conservative substitutions), insertions or deletions, but anti-MCSP antibodies comprising this sequence retain the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:35. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the heavy chain sequence of SEQ ID NO: 35, including post-translational modifications of the sequence.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, and the amino acid sequence SEQ ID NO: Light chain of 97%, 98%, 99% or 100% sequence identity. In certain embodiments, a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence (eg, conservative substitutions), insertions or deletions, but anti-MCSP antibodies comprising this sequence retain the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO 34. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the light chain sequence of SEQ ID NO: 34, including post-translational modifications of the sequence.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises a heavy chain as in any of the embodiments provided above and any of the embodiments as provided above The light chain in the example. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 35 and a light chain sequence comprising the amino acid sequence SEQ ID NO: 34, including post-translational modifications of the sequences.

In another aspect, the anti-MCSP antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence SEQ ID NO:37. , 99% or 100% sequence identity heavy chain sequence. In certain embodiments, a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence (eg, conservative substitutions), insertions or deletions, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO:37. In certain embodiments, substitutions, insertions, or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the heavy chain sequence of SEQ ID NO: 37, including post-translational modifications of the sequence.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the amino acid sequence SEQ ID NO: Light chain of 97%, 98%, 99% or 100% sequence identity. In certain embodiments, a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity has a substitution relative to a reference sequence (eg, conservative substitutions), insertions or deletions, but the anti-MCSP antibody comprising the sequence retains the ability to bind to MCSP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO 36. In some embodiments In place, substitutions, insertions or deletions occur in regions other than the HVR (ie, in the FR). Optionally, the anti-MCSP antibody comprises the light chain sequence of SEQ ID NO: 36, including post-translational modifications of the sequence.

In another aspect, an anti-MCSP antibody is provided, wherein the antibody comprises a heavy chain as in any of the embodiments provided above and a light chain in any of the embodiments provided above. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 37 and a light chain sequence comprising the amino acid sequence SEQ ID NO: 36, including post-translational modifications of the sequences. In another aspect, the invention provides an antibody that binds to the same epitope as an anti-MCSP antibody provided herein.

In one embodiment, an antibody that binds to the same epitope as an anti-MCSP antibody having a VH comprising the amino acid sequence SEQ ID NO: 27 and a VL comprising the amino acid sequence SEQ ID NO: 26 is provided. In another embodiment, an antibody that binds to the same epitope as an anti-MCSP antibody having a VH comprising the amino acid sequence SEQ ID NO: 32 and a VL comprising the amino acid sequence SEQ ID NO: 31 is provided.

In other embodiments, antibodies that compete for binding to the same epitope as the anti-MCSP antibodies described herein are provided.

In one embodiment, an antibody that binds to the same epitope as an anti-MCSP antibody and/or competes for binding to the same epitope exhibits an effector functional activity, such as Fc-mediated cytotoxicity (including ADCC activity).

In one embodiment, the anti-MCSP antibody binds to a membrane proximal epitope of human MCSP. In one embodiment, the anti-MCSP antibody binds to a membrane proximal epitope of a human MCSP comprising a domain comprising a CSPG repeat. In one implementation In one embodiment, the anti-MCSP antibody binds to a membrane proximal epitope of human MCSP, the proximal epitope of the membrane being derived from the amino acid sequence SEQ ID NO: 5, located within or overlapping the sequence. In one embodiment, the anti-MCSP antibody binds to a membrane proximal epitope of human MCSP, the proximal epitope of the membrane being derived from the amino acid sequence SEQ ID NO: 4, located within or overlapping the sequence. In one embodiment, the anti-MCSP antibody binds to a membrane proximal epitope of human MCSP, the proximal epitope of the membrane being from the amino acid sequence SEQ ID NO: 3, located within or overlapping the sequence.

In another aspect of the invention, the anti-MCSP anti-system monoclonal antibody of any of the above embodiments comprises a chimeric, humanized or human antibody. In one embodiment, an anti-MCSP anti-system antibody fragment, such as an Fv, Fab, Fab', scFv, diabody or F(ab') ₂ fragment. In another embodiment, an anti-systemic full length antibody, such as an intact IgGl antibody or other antibody class or isotype as defined herein.

In one embodiment, the anti-MCSP anti-system mouse monoclonal antibody LC007. The nucleic acid sequences of the heavy and light chains of this antibody are found in SEQ ID NOs: 37 and 36, respectively. In one embodiment, the anti-MSCP anti-system is derived from a chimeric antibody to mouse monoclonal antibody LC007. In one embodiment, the anti-MSCP anti-system is derived from a humanized antibody to mouse monoclonal antibody LC007. In one embodiment, the anti-MSCP anti-system is derived from a human antibody to mouse monoclonal antibody LC007.

In another aspect, the anti-MCSP antibodies of any of the above embodiments can be incorporated into any of the features described in Sections 1-7 below, alone or in combination:

Antibody affinity

In certain embodiments, the dissociation constant (Kd) of an antibody provided herein 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM (eg 10 ^-8 M or lower, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M).

In one embodiment, the Kd is measured by radiolabeled antigen binding assay (RIA) performed using the Fab format of the antibody of interest and its antigen as described in the assay below. The solution binding affinity of the Fab to the antigen is measured by balancing the Fab with the lowest concentration of ( ^125I ) labeled antigen in the presence of an unlabeled antigen in the titration series, and then capturing it with an anti-Fab antibody coated plate. The antigen bound (see, for example, Chen et al, J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the analysis of the porous plate MICROTITER ^® (Thermo Scientific) stored in 50 mM sodium carbonate used (pH 9.6) in the 5 μg / ml of a capturing anti-Fab antibody (Cappel Labs) coated overnight and subsequently Block with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (about 23 ° C). In a non-adsorbing plate (Nunc 269620), 100 pM or 26 pM [ ¹²⁵ I] antigen is mixed with serial dilutions of the Fab of interest (for example, with Presta et al, Cancer Res. 57:4593-4599 (1997). The evaluation of anti-VEGF antibody (Fab-12) was consistent). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (eg, 1 hour). Solution was then removed and stored in PBS with 0.1% of polysorbate 20 (TWEEN-20 ^®) The plates were washed 8 times. When the dried plate, add 150 μl / scintillator (MICROSCINT-20 ^TM; Packard) of the hole, and the plate (Packard) counted on a TOPCOUNT ^TM γ counter 10 min. Concentrations with a maximum binding of less than or equal to 20% obtained for each Fab were selected for competitive binding assays.

According to another embodiment, an immobilized antigen of about 10 reaction units (RU) was used at 25 ° C using surface plasmon resonance analysis using BIACORE ^® -2000 or BIACORE ^® -3000 (BIAcore, Piscataway, NJ) The CM5 wafer is used to measure Kd. Briefly, N -ethyl- N' -(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) were used according to the supplier's instructions. ) to activate a carboxymethylated dextran biosensor wafer (CM5, BIACORE). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl/min to obtain about 10 reaction units (RU) of the coupled protein. After the antigen injection, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, at 25 deg.] C to about 25 μl / min of the flow rate of injection present in the Fab & 0.05% polysorbate 20 (TWEEN-20 ^TM) surfactant twice of PBS (PBST) containing ester in the serially diluted (0.78 nM to 500 nM). Association rate (k _on) and dissociation rates (k _off) system using a simple one Langmuir binding model (BIACORE ^® Evaluation Software version 3.2) by simultaneous fitting the association and the solution is calculated from the sensed FIG. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off /k _on . See, for example, Chen et al, J. Mol. Biol. 293:865-881 (1999). If the on-rate obtained by the above surface plasmon resonance analysis exceeds 10 ⁶ M ^-1 s ^-1 , the association rate can be determined by using a fluorescence quenching technique. Fluorescence emission intensity of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) measured at 25 ° C in the presence of increasing concentrations of antigen (excitation = 295 nm; emission = 340 nm, 16 nm) bandpass) of raising or lowering, as spectrometers use than stirring the color of the tube (e.g., a spectrometer equipped with a (Aviv Instruments equipment stopped flow) or a 8000-series SLM-AMINCO ^TM spectrophotometer (ThermoSpectronic)) as measured.

2. Antibody fragment

In certain embodiments, anti-system antibody fragments are provided herein. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün , The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore ed. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185 ; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising a salvage receptor binding epitope residue and an in vivo half-life extension, see U.S. Patent No. 5,869,046.

A double-stranded anti-system has two antigen-binding sites which are bivalent or bispecific antibody fragments. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) . Tri-chain antibodies and four-chain antibodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).

A single domain anti-system comprises antibody fragments that comprise all or a portion of a heavy chain variable domain or all or a portion of a light chain variable domain in an antibody. In certain embodiments, a single domain anti-system human single domain antibody (Domantis, Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).

Antibody fragments can be produced by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, an anti-system chimeric antibody is provided herein. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (eg, a monkey)) and a human constant region. In another example, an antibody to a "class switching" of a chimeric anti-system, wherein the class or subclass has been altered relative to the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric anti-system humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein the HVR (eg, a CDR, or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may optionally comprise at least a portion of a human constant region. In some embodiments, some of the FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody that produces an HVR residue) to, for example, restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and further described, for example, in Riechmann et al, Nature 332:323- 329 (1988); Queen et al . Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Explaining SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (explaining "surface remodeling");Dall'Acqua et al., Methods 36:43-60 (2005) (Explaining "FR Reorganization"); and Osbourn et al, Methods 36: 61-68 (2005) and Klimka et al, Br . J. Cancer , 83: 252-260 (2000) ( Explain the "guided selection" method of FR restructuring.

The human framework area that can be used for humanization includes (but is not limited to): the framework area selected using the "best fit" method (see, for example, Sims et al. , J. Immunol. 151:2296 (1993)); a framework region of a consensus sequence of human antibodies of a particular subgroup of a chain or heavy chain variable region (see, eg, Carter et al, Proc. Natl. Acad. Sci. USA , 89: 4285 (1992); and Presta et al, J. Immunol. , 151: 2623 (1993)); human maturation ( transformation ) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); And framework regions obtained from screening FR libraries (for example, see Baca et al, J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al, J. Biol. Chem. 271: 22611-22618 (1996) ).

4. Human antibodies

In certain embodiments, anti-system human antibodies are provided herein. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol . 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol . 20: 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is extrachromosomally or randomly integrated into an animal chromosome. In these transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). For example, See also U.S. Patent No. 6,075,181 and No. 6,150,584, which describes XENOMOUSE ^TM technologies; U.S. Patent No. 5,770,429, which describes techniques HUMAB®; U.S. Patent No. 7,041,870, which describes techniques KM MOUSE®; and U.S. Patent Application Publication Case US 2007/0061900, which describes VELOCIMOUSE® technology. Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be produced by fusion-based methods. Human myeloma and mouse-human interspecies myeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol. , 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications , pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J .Immunol. , 147: 86 (1991). Human antibodies produced by human B cell fusion tumor technology are also described in Li et al, Proc. Natl. Acad. Sci. USA , 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Patent No. 7,189,826 (which describes the production of a single human IgM antibody from a fusion tumor cell line) and Ni, Xiandai Mianyixue , 26(4): 265-268 (2006). Human fusion tumor). Human fusion tumor technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology , 20(3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies can also be produced by isolating Fv pure line variable domain sequences selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are set forth below.

5. Antibodies derived from the library

An antibody of the invention can be isolated by screening a combinatorial library for antibodies having the desired activity. For example, a variety of methods are known in the art for producing phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, 2001) and further described, for example, in the following literature Middle: McCafferty et al, Nature 348:552-554; Clackson et al, Nature 352:624-628 (1991); Marks et al, J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J .Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004).

In some phage display methods, VH and VL gene profiles are separately selected by polymerase chain reaction (PCR) and randomly recombined in phage libraries, and antigen-binding phage, such as Winter, can then be screened in such libraries. Et al., Ann. Rev. Immunol . , 12: 433-455 (1994). Phage typically display a single-chain Fv (scFv) fragment or an antibody fragment that is a Fab fragment. Libraries from immunized sources provide high affinity antibodies against the immunogen and do not require the construction of fusion tumors. Alternatively, the native profile (e.g., from humans) can be selected without any immunization to provide a single antibody source for a wide variety of non-autoantigen and autoantigens, such as Griffiths et al, EMBO J, 12: 725-734 ( 1993). Finally, a natural library can also be produced synthetically by selecting a non-rearranged V-gene fragment from a stem cell and using a PCR primer containing a random sequence to encode a highly variable CDR3 region and rearranging in vitro. As described by Hoogenboom and Winter, J. Mol. Biol. , 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007 /0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

An antibody or antibody fragment isolated from a human antibody library is considered herein to be a human antibody or a human antibody fragment.

6. Multispecific antibodies

In certain embodiments, anti-system multispecific antibodies, such as bispecific antibodies, are provided herein. A multispecific antibody that has binding specificity for at least two different sites. In certain embodiments, one binding specificity is for MCSP and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of MCSP. Cytotoxic agents can also be concentrated to cells expressing MCSP using bispecific antibodies. Bispecific antibodies can be made in the form of full length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinantly displaying two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/ 08829 and Traunecker et al., EMBO J. 10:3655 (1991)) and "knob-in-hole" modification (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering an electrostatic traction effect for the preparation of antibody Fc-heterodimeric molecules (WO 2009/089004 A1); cross-linking two or more antibodies or fragments (eg See, U.S. Patent No. 4,676,980 and Brennan et al, Science 229:81 (1985); the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al, J. Immunol. , 148(5): 1547 -1553 (1992)); "Double-chain antibody" technology for the preparation of bispecific antibody fragments (see, for example, Hollinger et al, Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)) And using single-chain Fv (scFv) dimers (for example, see Gruber et al, J. Immunol. 152: 5368 (1994)); and preparing trispecific antibodies (for example, such as Tutt et al. , J. Immunol. 147:60 (1991)).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "Octopus antibodies" (see, for example, US 2006/0025576 A1).

An antibody or fragment herein also includes "dual acting FAb" or "DAF" comprising an antigen binding site that binds to MCSP and another different antigen (see, for example, US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications in the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct has the desired characteristics (eg, antigen binding).

a) substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. The conservative substitutions are shown under the heading "Conservative substitutions" in Table 1. Other substantial changes are shown under the heading "Example Substitutions" in Table 1, and are further described below with reference to the amino acid side chain classes. Amino acid substitution can be introduced into the antibody of interest and In the products screened for the desired activity (eg, maintained/improved antigen binding, reduced immunogenicity or modified ADCC or CDC).

Amino acids can be grouped according to common side chain properties: (1) hydrophobicity: n-leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions must exchange members of one of these categories for another category.

One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variants selected for further study will alter (eg, improve) certain biological properties (eg, increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain the parent antibody. Certain biological properties. Exemplary substituted system affinity matured antibodies, which can be conveniently produced, for example, using phage-displayed affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for specific biological activities (eg, binding affinity).

HVRs can be altered (eg, substituted) to, for example, improve antibody affinity. HVR "hot spots" (ie, residues encoded by codons that mutate at high frequencies during somatic cell maturation, for example, see Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / These changes were made in SDR (a-CDR) and the binding affinity of the resulting variant VH or VL was tested. Affinity maturation by constructing secondary libraries and reselection from such libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., edited by Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis) In the gene. Then create a secondary library. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs as long as such alterations do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) can be performed at the HVR without significantly reducing binding affinity. These changes can be located outside of the HVR "hotspot" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered or contains no more than one, two or three amino acid substitutions.

A method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science , 244: 1081-1085. In this method, one of the residues or groups of residues in the target residue (eg, charged residues such as arg, asp, his, lys, and glu) is identified and neutralized by a neutral or negatively charged amino acid ( For example, alanine or polyalanine) is substituted to determine if it affects the interaction of the antibody with the antigen. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact point between the antibody and the antigen is identified by the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or excluded as replacement candidates. Variants can be screened to determine if they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions (lengths ranging from one residue to polypeptides containing hundreds or more residues) and sequences of single or multiple amino acid residues insert. Examples of terminal insertions include antibodies having N-terminal methylthioguanidine residues. Other insertional variants of the antibody molecule include an N-terminal or C-terminal fusion of the antibody with an enzyme that extends the serum half-life of the antibody (eg, for ADEPT) or a polypeptide.

b) glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. An increase or a deletion of a glycosylation site in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody comprises an Fc region, the carbohydrate to which it is attached may vary. Native antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide, which is typically attached to Asn297 of the CH2 domain in the Fc region by an N-linkage. See, for example, Wright et al, TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates such as mannose, N-ethyl glucosamine (GlcNAc), galactose, and sialic acid, and the algae of GlcNAc attached to the "backbone" of the biantennary oligosaccharide structure. sugar. In some embodiments, an oligosaccharide in an antibody of the invention can be modified to produce an antibody variant having certain improved properties.

In one embodiment, antibody variants lacking fucose attached (directly or indirectly) to the Fc region are provided in the carbohydrate structure. For example, the amount of fucose in the antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (eg, complex, heterozygous, and high mannose structures), such as Measured by MALDI-TOF mass spectrometry as described, for example, in WO 2008/077546. Asn297 refers to an aspartate residue located at about 297 (Eu number of the Fc region residue) in the Fc region; however, Asn297 may also be located upstream or downstream of 297 due to minor sequence changes in the antibody. The three amino acids are between 294 and 300. Such fucosylated variants can have improved ADCC function. See, for example, U.S. Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Corporation). Examples of publications relating to "defucosylation" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/ US Patent Publication No. 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; 053742; WO 2002/031140; Okazaki et al, J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al, Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al, Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al, especially in Example 11) and gene knockout cell lines (eg α-1,6-fucosyltransferase gene FUT8) Excluded CHO cells, for example, see Yamane-Ohnuki, Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng. , 94(4): 680-688 (2006); 2003/085107).

Further provided are antibody variants having a bisecting oligosaccharide wherein, for example, the biantennary oligosaccharide attached to the Fc region of the antibody is halved by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.), WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.).

Accordingly, the invention further relates to a method for improving the glycosylation profile of an anti-MCSP antibody of the invention produced by a host cell comprising expressing a nucleic acid encoding an anti-MCSP antibody of the invention in the host cell and encoding a glycosyl group A nucleic acid that transfers a polypeptide of the enzyme activity or a vector comprising the nucleic acid. Genes having glycosyltransferase activity include β(1,4)-N-acetylglucosamine transferase III (GnTII), α-mannosidase II (ManII), β(1,4)-galactosyl Transferase (GalT), β(1,2)-N-acetylglucosamine transferase I (GnTI) and β(1,2)-N-acetylglucosamine transferase II (GnTII). In one embodiment, a combination of genes having glycosyltransferase activity (eg, GnTIII and Man II) is expressed in a host cell. Likewise, the method also encompasses the expression of one or more polynucleotides encoding an anti-MCSP antibody in a host cell, In the main cell, the glycosyltransferase gene has been disrupted or otherwise inactivated (for example, in a host cell, the activity of the gene encoding the α1-6 core fucosyltransferase has been eliminated). In another embodiment, an anti-MCSP antibody of the invention can be produced in a host cell that additionally expresses a polynucleotide encoding a polypeptide having GnTIII activity to improve glycosylation patterns. In a specific embodiment, the polypeptide having GnTIII activity is a fusion polypeptide comprising a Golgi localization domain of a Golgi resident polypeptide. The term Golgi localization domain refers to an amino acid sequence in the Golgi resident polypeptide responsible for anchoring the polypeptide in the Golgi complex. Typically, the localization domain comprises the amino terminus "tail" of the enzyme. In another preferred embodiment, the anti-MCSP antibody of the invention exhibits an anti-MCSP with enhanced Fc receptor binding affinity and enhanced effector function in a host cell which exhibits a polynucleotide encoding a polypeptide having GnTIII activity. antibody. Thus, in one embodiment, the invention relates to a host cell comprising (a) an isolated nucleic acid comprising a sequence encoding a polypeptide having GnTIII activity; and (b) an isolated polynucleotide encoding the anti-invention of the invention MCSP antibodies, such as chimeric, primatized or humanized antibodies that bind to human MCSP. In a preferred embodiment, the polypeptide having GnTIII activity comprises a fusion polypeptide of the catalytic domain of GnTIII and the Golgi localization domain is the localization domain of mannosidase II. The method of producing such fusion polypeptides and using the same to produce an effector-enhancing antibody is disclosed in U.S. Provisional Patent Application Serial No. 60/495,142, and U.S. Patent Application Publication No. 2004/0241817, the entire contents of which are expressly incorporated by reference. Incorporated herein. In a specific embodiment, the modified anti-MCSP antibody produced by the host cell has an IgG constant region or comprises an Fc region thereof Fragment. In another specific embodiment, the anti-MCSP anti-systematic antibody or a fragment thereof comprising an Fc region.

An anti-MCSP antibody with altered glycosylation produced by a host cell of the invention typically exhibits enhanced Fc receptor binding affinity and/or enhancement effects due to improved host cells (eg, due to the expression of a glycosyltransferase gene). Subfunction. Preferably, the enhanced Fc receptor binding affinity enhances binding to an Fc[gamma] activating receptor (e.g., FcyRIIIa receptor). The enhanced effector function is preferably enhanced by one or more of the following: enhanced antibody-dependent cellular cytotoxicity, enhanced antibody-dependent cellular phagocytosis (ADCP), enhanced interleukin secretion, enhanced immune complexes Mediated antigen uptake by antigen presenting cells, enhanced Fc-mediated cytotoxicity, enhanced binding to NK cells, enhanced binding to macrophages, enhanced binding to polymorphonuclear cells (PMN), Enhanced binding to mononuclear spheres, enhanced target-binding antibody cross-linking, enhanced direct signal transduction of apoptosis, enhanced dendritic cell maturation, and enhanced T cell priming.

In one aspect, the invention provides an anti-MCSP antibody (eg, a variant antibody) having enhanced effector function (including antibody-dependent cellular cytotoxicity) compared to an anti-MCSP antibody that has not been glycosylated. Glycoform. Glycosylation modifications of antibodies have been previously described. See, for example, U.S. Patent No. 6,602,684, hereby incorporated by reference herein in entirety Methods for producing anti-MCSP antibodies from host cells with altered gene activity involved in glycosylation are also described in detail herein (see, for example, the previous section entitled "Expression Vectors and Host Cells"). The enhancement of ADCC of the anti-MCSP antibody of the present invention is also enhanced by affinity of the antibody for MCSP. (For example, by affinity maturation or other methods of improving affinity (see Tang et al., J. Immunol. 2007, 179: 2815-2823)). The invention also encompasses combinations of such methods.

Recently, clinical trials of unconjugated monoclonal antibodies (mAbs) for the treatment of some types of cancer have yielded encouraging results. Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997); Deo et al., Immunology Today 18: 127 (1997). Chimeric unconjugated IgG1 has been approved for use in low-grade or follicular B-cell non-Hodgkin's lymphoma, Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997); and another unconjugated mAb (Humanized IgG1 targeting solid breast tumors) has also shown promising results in Phase III clinical trials, Deo et al., Immunology Today 18: 127 (1997). The antigens of the two mAbs are expressed in large amounts in their tumor cells, respectively, and these antibodies mediate potential tumor destruction by effector cells in vitro and in vivo. In contrast, many other unconjugated mAbs with good tumor specificity do not trigger effector functions with potent clinically useful potential. Frost et al, Cancer 80:317-33 (1997); Surfus et al, J. Immunother. 19: 184-91 (1996). For some of these weaker mAbs, adjuvant interleukin therapy is currently being tested. The addition of interleukins stimulates antibody-dependent cellular cytotoxicity (ADCC) by increasing the activity and number of circulating lymphocytes. Frost et al, Cancer 80:317-33 (1997); Surfus et al, J. Immunother. 19: 184-91 (1996). ADCC (cleavage attack on targeted cells) is triggered after binding of the white blood cell receptor to the constant region (Fc) of the antibody. Deo et al., Immunology Today 18: 127 (1997).

A different but complementary method of increasing the ADCC activity of unconjugated IgGl is to engineer the Fc region of the antibody. Protein engineering studies have shown that FcyR interacts with the lower hinge region of the IgG CH2 domain. Lund et al., J. Immunol. 157:4963-69 (1996). However, FcγR binding also requires the presence of oligosaccharides covalently attached to the conserved Asn 297 in the CH2 region, Lund et al, J. Immunol. 157:4963-69 (1996); Wright and Morrison, Trends Biotech. :26-31 (1997), indicating that both oligosaccharides and polypeptides directly promote the site of interaction, or that oligosaccharides are required to maintain the conformation of the active CH2 polypeptide. Therefore, modifications to the structure of the oligosaccharide can be explored as a means of increasing the affinity of the interaction.

The IgG molecule carries two N-linked oligosaccharides in its Fc region, one for each heavy chain. As any glycoprotein, antibodies are produced in a glyco-type population that share the same polypeptide backbone but have different oligosaccharides attached to the glycosylation site. Oligosaccharide complex biantenna type commonly found in the Fc region of serum IgG (Wormald et al, Biochemistry 36: 130-38 (1997)), which has low levels of terminal sialic acid and bisected N-acetamidine Glucosamine (GlcNAc), as well as varying degrees of terminal galactosylation and core fucosylation. Some studies have shown that the minimal carbohydrate structure required for FcγR binding is located within the oligosaccharide core. Lund et al., J. Immunol. 157:4963-69 (1996).

Mouse or hamster-derived cell lines used in industry and academia to generate unconjugated therapeutic mAbs typically attach the desired oligosaccharide determinant to the Fc site. However, the IgGs expressed in these cell lines lack the bisected GlcNAc found in small amounts in serum IgG. Lifely et al, Glycobiology 318: 813-22 (1995). In contrast, it has recently been observed that humanized IgG1 (CAMPATH-1H) produced by rat myeloma carries a halved GlcNAc in some of its glycosylation patterns. Lifely et al, Glycobiology 318: 813-22 (1995). Rat cell-derived antibodies achieve maximum in vitro ADCC activity similar to the CAMPATH-1H antibody produced in standard cell lines, but antibody concentrations are significantly lower.

The CAMPATH antigen is usually present in large amounts on lymphoma cells, and this chimeric mAb has high ADCC activity in the absence of the aliquot of GlcNAc. Lifely et al, Glycobiology 318: 813-22 (1995). In the N-linked glycosylation pathway, a bisected GlcNAc is added by GnTIII. Schachter, Biochem. Cell Biol. 64: 163-81 (1986).

Previous studies have used a single CHO cell line that produces antibodies that have been previously engineered to exhibit varying degrees of GnTIII enzyme gene in an externally regulated manner (Umaña, P., et al, Nature Biotechnol. 17: 176-180 (1999)). . This method establishes for the first time a strict correlation between the performance of a glycosyltransferase (eg, GnTIII) and the ADCC activity of a modified antibody. Thus, the invention encompasses an anti-MCSP antibody comprising an Fc region or a region equivalent to an Fc region having altered glycosylation by altering the degree of expression of a glycosyltransferase gene in a host cell producing the antibody . In a specific embodiment, the change in the degree of gene expression is an increase in GnTIII activity. In the Fc region of the antibody, increased GnTIII activity increases the percentage of diploid oligosaccharides and decreases the percentage of fucose residues. This antibody or fragment thereof has enhanced Fc receptor binding affinity and enhanced effector function.

The invention also relates to the production of anti-MCSP having modified oligosaccharides of the invention A method of antibody comprising (a) cultivating a host cell engineered to exhibit at least one nucleic acid encoding a polypeptide having glycosyltransferase activity under conditions permitting production of an anti-MCSP antibody of the invention, wherein the glycosyltransferase The active polypeptide is expressed in an amount sufficient to modify the oligosaccharide in the Fc region of the anti-MCSP antibody produced by the host cell; and (b) isolating the anti-MCSP antibody. In one embodiment, the polypeptide having glycosyltransferase activity is GnTIII. In another embodiment, there are two polypeptides having glycosyltransferase activity. In a particular embodiment, two peptides having glycosyltransferase activity are GnTIII and ManII. In another embodiment, the polypeptide having glycosyltransferase activity is a fusion polypeptide comprising a catalytic domain of GnTIII. In another specific embodiment, the fusion polypeptide additionally comprises a Golgi localization domain of the Golgi resident polypeptide. Preferably, the Golgi localization domain is a localization domain of mannosidase II or GnTI. Alternatively, the Golgi localization domain is selected from the group consisting of a localization domain of mannosidase I, a localization domain of GnTII, and a localization domain of an alpha 1-6 core fucosyltransferase. The anti-MCSP antibodies produced by the methods of the invention have enhanced Fc receptor binding affinity and/or enhanced effector function. Typically, the enhanced effector function is one or more of the following: enhanced Fc-mediated cytotoxicity (including enhanced antibody-dependent cellular cytotoxicity), enhanced antibody-dependent cellular phagocytosis (ADCP), enhanced cells Interleukin secretion, enhanced immune complex-mediated antigen uptake by antigen-presenting cells, enhanced binding to NK cells, enhanced binding to macrophages, enhanced binding to mononuclear spheres, enhanced and more Binding of nucleated cells, enhanced direct signal transduction of apoptosis, enhanced target-binding antibody cross-linking, enhanced dendritic cells Mature or enhanced T cell priming. Enhanced Fc receptor binding affinity is preferably enhanced by binding to an Fc activating receptor (e.g., FcyRIIIa). In a particularly preferred embodiment, the ABM is a humanized antibody or fragment thereof.

In one embodiment, the percentage of the aliquoted N-linked oligosaccharide in the Fc region of the anti-MCSP antibody is at least about 10% to about 100%, specifically at least about 50%, more specifically the total oligosaccharide. At least about 60%, at least about 70%, at least about 80%, or at least about 90-95%. In another embodiment, the antibody produced by the methods of the invention has an elevated proportion of non-fucosylated oligosaccharides in the Fc region due to modification of its oligosaccharides by the methods of the invention. In one embodiment, the percentage of non-fucosylated oligosaccharides is at least about 20% to about 100%, specifically at least about 50%, at least about 60% to about 70%, and more specifically at least about 75%. Non-fucosylated oligosaccharides can be heterozygous or complex types. In another embodiment, the antibody produced by the methods of the invention has an elevated ratio of aliquoted oligosaccharides in the Fc region by modification of its oligosaccharides by the methods of the invention. In one embodiment, the percentage of bipartite oligosaccharides is at least about 20% to about 100%, specifically at least about 50%, at least about 60% to about 70%, and more specifically at least about 75%. . In a particularly preferred embodiment, the anti-MCSP antibody produced by the host cell and the method of the invention has an elevated proportion of the bisected non-fucosylated oligosaccharide in the Fc region. The bisected non-fucosylated oligosaccharides can be heterozygous or complex types. In particular, the methods of the invention can be used to produce antibodies wherein at least about 10% to about 100%, specifically at least about 15%, more specifically at least about 20% to about 50%, in the Fc region of the antibody. More specifically, at least about 20% to about 25%, and more specifically at least about 30% to about 35%, of the oligosaccharide bipartite non-fucosylated oligosaccharides. The anti-MCSP antibodies of the invention may also comprise an Fc region, wherein in the Fc region of the anti-MCSP antibody, at least about 10% to about 100%, specifically at least about 15%, more specifically at least about 20% to About 25%, and more specifically at least about 30% to about 35%, of the oligosaccharide bipartite heterozygous non-fucosylated oligosaccharides.

In another embodiment, the invention relates to an anti-MCSP antibody produced by the methods of the invention, which is engineered to have enhanced effector function and/or enhanced Fc receptor binding affinity. Enhanced effector functions can include, but are not limited to, one or more of: enhanced Fc-mediated cytotoxicity (including enhanced antibody-dependent cellular cytotoxicity), enhanced antibody-dependent cellular phagocytosis (ADCP) Enhanced interleukin secretion, enhanced immune complex-mediated antigen uptake by antigen-presenting cells, enhanced binding to NK cells, enhanced binding to macrophages, enhanced binding to mononuclear cells, Enhanced binding to polymorphonuclear cells, enhanced direct signal transduction of apoptosis, enhanced target-binding antibody cross-linking, enhanced dendritic cell maturation or enhanced T cell priming. In a preferred embodiment, the enhanced Fc receptor binding affinity enhances binding to the Fc activating receptor (optimal FcyRIIIa). In one embodiment, the anti-system intact antibody. In one embodiment, the anti-system comprises an antibody fragment of the Fc region, or a fusion protein comprising a region equivalent to the Fc region of the immunoglobulin.

The invention further provides methods of producing and using a host cell system for producing a glycosyl form of an antibody of the invention having enhanced Fc receptor binding affinity (preferably enhanced binding to an Fc activating receptor) and / or have enhanced effector functions (including antibody-dependent cellular toxicity). Can be used in this hair The method of modifying the sugar of the antibody has been described in more detail in the following documents: U.S. Patent No. 6,602,684, U.S. Patent Application Publication No. 2004/0241817 A1, U.S. Patent Application Publication No. 2003/0175884 A1, Provisional U.S. Patent Application The entire contents of each of the patents are incorporated herein by reference in its entirety. Alternatively, an antibody of the invention may be glycosylated to have a reduced fucose residue in the Fc region according to the techniques disclosed in U.S. Patent Application Publication No. 2003/0157108 (Genentech) or EP 1 176 195. A1, WO 03/084570, WO 03/085119, and U.S. Patent Application Publication Nos. 2003/0115614, 2004/093621, 2004/110282, 2004/110704, and 2004/132140 (Kyowa). The contents of each of these documents are incorporated herein by reference in their entirety. The glycosylation-modified antibodies of the present invention can also be produced in a system for producing modified glycoproteins, for example, as taught in U.S. Patent Application Publication Nos. 60/344,169 and WO 03/056914 (GlycoFi). The company or WO 2004/057002 and WO 2004/024927 (Greenovation), each of which is incorporated herein in its entirety by reference.

In another aspect, the invention provides a host cell expression system for producing an antibody of the invention having a modified glycosylation pattern. In particular, the invention provides a host cell system of a glycosyl type having improved therapeutic value for use in producing an antibody of the invention. Accordingly, the invention provides a host cell expression system selected or engineered to exhibit a polypeptide having glycosyltransferase activity. In a specific embodiment, the glycosyltransferase activity is GnTIII activity. In one embodiment, the polypeptide having GnTIII activity comprises a heterologous Golgi resident A fusion polypeptide of the Golgi localization domain of the polypeptide. In particular, the host cell expression systems can be engineered to comprise a recombinant nucleic acid molecule encoding a polypeptide having GnTIII operably linked to a constitutive or regulated promoter system.

In a specific embodiment, the invention provides a nucleic acid in which a host cell has been engineered to exhibit at least one fusion polypeptide encoding a Golgi localization domain having GnTIII activity and comprising a heterologous Golgi resident polypeptide. In one aspect, the host cell is engineered by a nucleic acid molecule comprising at least one gene encoding a fusion polypeptide having GnTIII activity and comprising a Golgi localization domain of a heterologous Golgi resident polypeptide.

Generally, any type of cultured cell line (including the cell lines discussed above) can be used as a background for engineering a host cell line of the invention. In a preferred embodiment, CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or fusion tumor cells, other mammals are used. Cells, yeast cells, insect cells or plant cells serve as background cells for the production of engineered host cells of the invention.

The invention is intended to encompass any engineered host cell that exhibits a polypeptide having glycosyltransferase activity (eg, GnTIII activity), including a fusion polypeptide comprising a Golgi localization domain of a heterologous Golgi resident polypeptide as defined herein.

One or several nucleic acids encoding a polypeptide having glycosyltransferase activity (eg, GnTIII activity) can be expressed under the control of a constitutive promoter or a regulatory expression system. Such systems are well known in the art and include the discussion above The system. If the host cell system comprises several different nucleic acids encoding a fusion polypeptide having glycosyltransferase activity (eg, GnTIII activity) and comprising a Golgi localization domain of a heterologous Golgi resident polypeptide, some of the nucleic acids It can be expressed under the control of a constitutive promoter, while other nucleic acids are expressed under the control of a regulated promoter. The degree of expression of a fusion polypeptide having glycosyltransferase activity (e.g., GnTIII activity) is determined by methods known in the art, including Western blot analysis, Northern blot analysis, and reporting. Subgene expression analysis or measurement of glycosyltransferase activity (eg, GnTIII activity). Alternatively, a lectin that binds to a biosynthetic product of GnTIII, such as E4-PHA lectin, may be employed. Alternatively, functional assays can be used which measure enhanced Fc receptor binding or enhanced effector function by antibodies produced by nucleic acid engineered cells encoding polypeptides having glycosyltransferase activity (eg, GnTIII activity). .

Host cells containing the coding sequences of the antibodies of the invention and which exhibit biologically active gene products can be identified by at least four general methods: (a) DNA-DNA or DNA-RNA hybridization; (b) "marker" gene function Presence or absence; (c) assessing the degree of transcription as measured by the performance of individual mRNA transcripts in the host cell; and (d) as measured by immunoassay or by biological activity of the gene product To detect these gene products.

In the first method, an anti-MCSP antibody can be detected by a DNA-DNA or DNA-RNA hybridization method using a probe comprising a nucleotide sequence homologous to a respective coding sequence or a portion or derivative thereof. The coding sequence and/or the coding sequence of a polypeptide having a glycosyltransferase (eg, GnTIII) activity in.

In the second method, based on certain "marker" gene functions (eg, thymidine kinase activity, antibiotic resistance, methotrexate resistance, transformed phenotype, inclusion formation in baculovirus, etc., etc.) The presence or absence of a recombinant expression vector/host system is identified and selected. For example, if the coding sequence of the antibody of the present invention or a fragment thereof and/or the coding sequence of a polypeptide having a glycosyltransferase (for example, GnTIII) activity is inserted into the marker gene sequence of the vector, the function of the marker gene can be utilized. It does not exist to identify recombinants containing the respective coding sequences. Alternatively, the marker gene can be placed in tandem with the coding sequence under the control of the same or different promoters used to control the expression of the coding sequence. The marker is indicative of the expression of the coding sequence of an antibody of the invention and/or the coding sequence of a polypeptide having glycosyltransferase (e.g., GnTIII) activity, depending on the expression of induction or selection.

In the third method, the transcriptional activity of the coding region of the antibody of the present invention or a fragment thereof and/or the coding sequence of a polypeptide having a glycosyltransferase (e.g., GnTIII) activity can be evaluated by a hybridization assay. For example, a probe homologous to a coding sequence of an antibody of the present invention or a fragment thereof and/or a polypeptide having a glycosyltransferase (eg, GnTIII) activity or a specific portion thereof can be used by the Northern blot method. To isolate RNA and analyze. Alternatively, the total nucleic acid of the host cell can be extracted and analyzed for hybridization to the probes.

In the fourth method, the performance of the protein product can be evaluated immunologically, for example, by Western blotting, immunoassays (e.g., radioimmunoprecipitation, enzyme-linked immunoassay, and the like). However, the final test for the success of the performance system involves the detection of biologically active gene products.

c) Fc region variant

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. An Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (eg, a substitution) at one or more amino acid positions (eg, an Fc region of human IgGl, IgG2, IgG3, or IgG4).

In certain embodiments, the invention encompasses antibody variants having some, but not all, effector functions, which renders the in vivo half-life of the antibody important, but certain effector functions (eg, complement and ADCC) are not necessary Or expected candidates for harmful applications. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/consumption of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding ability (and thus may lack ADCC activity), but retains FcRn binding ability. Primary cells (NK cells) used to mediate ADCC only display Fc (RIII, whereas monocytes exhibit Fc (RI, Fc (RII and Fc (RIII. FcR on hematopoietic cells are outlined in Ravetch and Kinet, Annu) Non-limiting examples of in vitro analysis to evaluate ADCC activity of a molecule of interest are set forth in U.S. Patent No. 5,500,362 (for example, see Hellstrom, pp. Immunol 9:457-492 (1991). I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); 5,821,337 (see Bruggemann, M, et al., J.Exp.Med 166:. 1351-1361 (1987 )). , or, non-radioactive analysis methods may be employed (e.g., the ACTI ^(TM) non-radioactive see for flow cytometry of cell toxicity assay (CellTechnology Corporation, Mountain View, CA) and CytoTox 96 ^® non-radioactive cytotoxicity Assay (Promega, Madison, WI)) . Useful effector cells for such analysis to include peripheral blood mononuclear cells (PBMC) and Natural killer (NK) cells. Or alternatively, may be in vivo (for example, in animal models, such as in Clynes et al., Proc. Nat'l Acad. S Ci. USA 95: 652-656 (1998)) Evaluation of ADCC activity of the molecule of interest. C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. See, for example, WO 2006/029879 And C1q and C3c binding ELISA in WO 2005/100402. To assess complement activation, CDC analysis can be performed (see, for example, Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996); Cragg, MS et al, Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determination can also be performed using methods known in the art (eg, see Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

A recognized in vitro ADCC assay is as follows: 1) the assay uses target cells known to represent the target antigen recognized by the antigen binding region of the antibody; 2) the assay uses humans isolated from blood of a randomly selected healthy donor Peripheral blood mononuclear cells (PBMC) were used as effector cells; 3) The assay was performed according to the following protocol: i) The PBMCs were isolated using standard density centrifugation procedures and suspended in RPMI cells at 5 x 106 cells/ml. In the medium; ii) the target cells are grown by standard tissue culture methods, harvested from the exponential growth phase with a viability greater than 90%, washed in RPMI cell culture medium, labeled with 100 microcuries of 51Cr, washed with cell culture medium. Twice and resuspend in cell culture medium at a density of 105 cells/ml; iii) transfer 100 μl of the above final target cell suspension to each well of a 96-well microtiter plate; iv) place the antibody in the cell The medium was serially diluted from 4000 ng/ml to 0.04 ng/ml and 50 μl of the resulting antibody solution was added to the target cells in a 96-well microtiter plate to test each antibody in the above concentration range in triplicate. Concentration; v) For the maximum release (MR) control, the other 3 wells containing labeled target cells received 50 μl of a 2% (V/V) aqueous solution of nonionic detergent (Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above); vi) For the spontaneous release (SR) control, the other 3 wells containing labeled target cells received 50 μl of RPMI cell culture medium instead of the antibody solution ( Iv point above); vii) then centrifuge the 96-well microtiter plate at 50 x g for 1 minute and at 4 ° C for 1 hour; viii) add 50 μl of PBMC suspension (point i above) To each well to obtain a 25:1 effector: target cell ratio, and place the plate in the incubator and hold for 4 hours at 37 ° C in a 5% CO ₂ atmosphere; ix) harvest cell-free supernatant from each well And quantify the radioactivity (ER) released by the experiment using a gamma counter; x) calculate the percentage of specific lysis at each antibody concentration according to the formula (ER-MR) / (MR-SR) × 100, where the ER is at the antibody concentration The average radioactivity quantified below (see point ix above), the average radioactivity quantified by the MR-based MR control (see point v above) (see point ix above), and the SR-series SR pair (see point vi above) quantified average radioactivity (see point ix above); 4) "enhanced ADCC" is defined as the percentage of maximum specific lysis observed over the range of antibody concentrations tested above. Increasing, and/or achieving a decrease in the half-desired antibody concentration, which is one of the maximum specific lysis rates observed over the range of antibody concentrations tested above. The enhancement of ADCC is relative to the ADCC mediated by the same antibody using the same assay, which is produced, purified, formulated and stored using the same standards known to those skilled in the art. Host cells are produced, but not by host cells engineered to express GnTIII.

Antibodies having reduced effector functions include those having substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of the amino acid positions 265, 269, 270, 297 and 327, including the substitution of alanine at residues 265 and 297. "DANA" Fc mutant (U.S. Patent No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are set forth. (See, for example, U.S. Patent No. 6,737,056, WO 2004/056312, and Shields et al, J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, the antibody variant comprises one or more modifications The Fc region of the amino acid substitution of ADCC (e.g., substitution at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region).

In some embodiments, the alteration in the Fc region alters (ie, ameliorates or decreases) C1q binding and/or complement dependent cytotoxicity (CDC), for example, as in U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al. , J. Immunol. 164: 4178-4184 (2000).

It has an extended half-life and enhanced neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgG to the fetus, Guyer et al, J. Immunol. 117:587 (1976) and Kim et al. , J. Immunol. 24 The antibody of the combination of 249 (1994)) is described in US 2005/0014934 A1 (Hinton et al.). These antibodies comprise a substituted Fc region having one or more modified Fc regions that bind to FcRn. The Fc variants include those having one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356 , 360, 362, 376, 378, 380, 382, 413, 424 or 434, such as the substitution of residue 434 of the Fc region (U.S. Patent No. 7,371,826).

See also, Duncan and Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648, 260; U.S. Patent No. 5,624,821; and WO 94/29351, to other examples of Fc region variants.

d) Cysteine-modified antibody variants

In certain embodiments, it may be desirable to produce a cysteine-engineered antibody, such as a "thio-mab", wherein one or more residues of the antibody are substituted with a cysteine residue. In a particular embodiment, the substituted residue is present at an accessible site of the antibody. Reactive sulfur by replacing their residues with cysteine The alcohol group is positioned at an accessible site of the antibody and can be used to couple the antibody to other moieties (eg, a drug moiety or a linker-drug moiety) to produce an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 of the heavy chain Fc region (EU number). The cysteine-modified antibody can be produced as described in, for example, U.S. Patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Portions suitable for derivatizing antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrole Pyridone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) And dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylated polyol (for example, glycerin), polyethylene Alcohols and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody is variable, and if more than one polymer is attached, it can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be modified, whether the antibody derivative will be used to define Treatment under conditions, etc.

In another embodiment, a conjugate of an antibody to a non-proteinaceous moiety is provided, which can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells, but can heat the non-proteinaceous portion to a temperature that kills cells in the vicinity of the antibody-non-proteinaceous portion.

B. Recombination methods and compositions

The antibodies can be produced using recombinant methods and compositions, as described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-MCSP antibody described herein is provided. The nucleic acid can encode an amino acid sequence comprising VL in the antibody and/or an amino acid sequence comprising VH (eg, a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (eg, expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (eg, has been transformed with such a substance): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH Or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NSO, Sp20 cells). In one embodiment, a method of making an anti-MCSP antibody is provided, wherein the method comprises, under conditions suitable for expressing the antibody The host cell comprising the nucleic acid encoding the antibody as provided above, and optionally the antibody is recovered from the host cell (or host cell culture medium).

For recombinant production of an anti-MCSP antibody, the nucleic acid encoding the antibody, as described above, is isolated and inserted into one or more vectors for further selection and/or expression in a host cell. The nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

Host cells suitable for use in the selection or expression of a vector encoding an antibody include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the performance of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology , Vol. 248 (BKCLo, ed., Humana a Press, Totowa, NJ, 2003), pp. 245-254, which illustrates the performance of antibody fragments in E. coli). After performance, the antibody can be isolated from the bacterial cell paste stored in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable colonization or expression hosts for vectors encoding antibodies, including glycosylation pathways that have been "humanized" to produce partially or fully human glycosylates. Fungi and yeast strains of the antibody of the model. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al, Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include Plants and insect cells. A variety of baculovirus strains have been identified for use in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. For example, see U.S. Pat. Nos. 5,959,177, No. 6,040,498, No. 6,420,548, No. 7,125,978 and No. 6,417,429 (describes antibodies generated PLANTIBODIES ^TM technology in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be used. Other examples of mammalian host cell lines that can be used are SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney line (293 or 293 cells, such as, for example, Graham et al, J. Gen Virol. 36:59 (1977) Said); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23: 243-251 (1980)); Monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human Hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells as described, for example, in Mather et al, Annals NYAcad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other available mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines, for example Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for the production of antibodies, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 ( 2003).

C. Analysis

The anti-MCSP antibodies provided herein can be identified by a variety of assays known in the art for screening or characterization of their physical/chemical properties and/or biological activity.

1. Combining analysis and other analysis

In one aspect, the antigen-binding activity of the antibody of the present invention is tested by a known method (e.g., ELISA, Western blotting, etc.).

In another aspect, competition assays can be used to identify antibodies that compete with the anti-MCSP antibodies described herein for binding to MCSP. In certain embodiments, such a competitive antibody binds to the same epitope (eg, a linear or conformational epitope) to which the anti-MCSP antibody described herein binds. A detailed exemplary method for mapping epitopes bound by antibodies is provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology , Vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized MCSP is grown in solution comprising a first labeled antibody that binds to MCSP and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to MCSP. The second antibody can be present in the supernatant of the fusion tumor. As a control, the immobilized MCSP was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to MCSP, excess unbound antibody is removed and the amount of label bound to immobilized MCSP is measured. If the amount of label bound to immobilized MCSP in the test sample is significantly reduced relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to MCSP. See Harlow and Lane (1988) Antibodies: A Laboratory Manual , Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity analysis

In one aspect, an assay for identifying a biologically active anti-MCSP antibody is provided. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the biological activity of an antibody of the invention is tested.

D. Immunoconjugate

The invention also provides immunoconjugates comprising one or more cytotoxic agents (chemotherapeutic agents or drugs, growth inhibitors, toxins (eg, protein toxins, bacterial, fungal, enzymatically active toxins of plant or animal origin or Its fragment) or radioisotope) is coupled to an anti-MCSP antibody herein.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoid (see US patent) No. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatin, such as the monomethyl auristatin drug moiety DE and DF (MMAE and MMAF, see U.S. Patents 5,635,483 and 5,780,588) And No. 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, Nos. 5,770,710, 5,773,001 and 5,877,296; Hinman et al, Cancer Res. 53:3336-3342 (1993); and Lode et al, Cancer Res. 58:2925-2928 (1998)); anthracycline antibiotics, For example, daunomycin or doxorubicin (see Kratz et al, Current Med. Chem. 13:477-523 (2006); Jeffrey et al, Bioorganic & Med . Chem. Letters 16:358-362 ( 2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005) Nagy et al, Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al, Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al, J. Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); Aminoguanidine; Vindesine; Taxanes, such as docetaxel, paclitaxel ), larotaxel, tesetaxel and ortataxel; trichothecene; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, diphtheria Non-binding active fragment of toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain , alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia Inhibitors, curcin, crotin, Sapaonaria officinalis inhibitors, Gelonin, mitogellin, restrictocin, phenomycin, enomycin, and crescent toxin.

In another embodiment, an immunoconjugate comprises an antibody described herein that is coupled to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When using a radioactive conjugate for detection, it may comprise a radioactive atom for scintillation studies, such as tc99m or I123; or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) For example, iodine-123 (ibid.), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, cesium, manganese or iron.

Conjugates of antibodies to cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as 3-(2-pyridyldithio)propionic acid N-succinimide (SPDP), 4-(N- Maleic iminomethyl)cyclohexane-1-carboxylic acid amber imidate (SMCC), imine cyclothiobutane (IT), bifunctional derivative of imidate (eg diimine) Dimethyl adipate HCl), active esters (eg diammonium iodide suberate), aldehydes (eg glutaraldehyde), bis-azido compounds (eg bis(p-azidobenzylidene) Hexamethylenediamine), bis-diazo derivatives (eg bis-(p-diazobenzimidyl)-ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate) and bis-active fluorine a compound (for example, 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987). Carbon-14-labeled 1-benzyl isothiocyanate-3-methyldiethylidamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for coupling radionucleotides to antibodies. See WO 94/11026. A linker can be a "cleavable linker" that promotes the release of a cytotoxic drug in a cell. For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker or a linker containing a disulfide bond can be used (Chari et al, Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

The immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared using, but not limited to, the following crosslinker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS , MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC and thio-SMPB and SVSB ((4-vinylindole) benzoic acid amber ylide), the above reagents are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., USA).

E. Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-MCSP antibodies provided herein can be used to detect the presence of MCSP in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative testing.

In one embodiment, an anti-MCSP antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of MCSP in a biological sample is provided. In certain embodiments, the methods comprise contacting a biological sample with an anti-MCSP antibody described herein under conditions permitting binding of the anti-MCSP antibody to MCSP, and detecting whether a complex is formed between the anti-MCSP antibody and the MCSP. The method can be an in vitro or in vivo method. In an embodiment In the mean, an anti-MCSP antibody is used to select a qualified individual for the therapy using an anti-MCSP antibody, for example, wherein the MCSP is used to select a biomarker for the patient.

Exemplary conditions that can be diagnosed using the antibodies of the invention include disorders characterized by the expression of MCSP, including cell proliferative disorders or angiogenic disorders. In one embodiment, the condition is cancer, such as skin cancer (including melanoma and basal cell carcinoma), glioma (including neutrophil blastoma), bone cancer (eg osteosarcoma), and leukemia (including ALL and AML) ).

In certain embodiments, a labeled anti-MCSP antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (eg, fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels, and radioactive labels) as well as indirectly detected via, for example, enzymatic or molecular interactions. (eg enzyme or ligand). Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores (eg, rare earth chelates or fluorescein and its derivatives), rose red (rhodamine) and its derivatives, dansyl, umbelliferone, luciferase (eg, firefly luciferase and bacterial luciferase, US Patent No. 4,737,456), fluorescent , 2,3-dihydropyridazinedione, wasabi peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (eg, glucose oxidase, Galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase, which are hydrogen peroxide with HRP, lactoperoxidase or microperoxidase) Enzymatic coupling of oxidation dye precursors), biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like.

F. Pharmaceutical formulations

A pharmaceutical formulation of an anti-MCSP antibody described herein is prepared by mixing the antibody of the desired purity with one or more pharmaceutically acceptable carriers ( Remington's Pharmaceutical Sciences , 16th Ed., Osol, A. Ed. (1980) ) is prepared as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methyl sulfide Aminic acid; preservative (for example, octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; p-hydroxybenzoic acid Alkyl esters such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10) Residue) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine, glutamic acid, aspartame, histamine Acid, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; Forming a counterion such as sodium; a metal complex (eg Zn-protein) Thereof); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary of the pharmaceutically acceptable carrier further herein, including interstitial drug dispersion agents, such as soluble neutral-active enzyme hyaluronic acid glycoprotein (a sHASEGP), such as a human soluble PH-20 enzyme hyaluronic acid glycoprotein, e.g. rHuPH20 (HYLENEX ^®, Baxter International). Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycanases (eg, chondroitinase).

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006/044908, the latter including a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient for the particular indication being treated, preferably with complementary agents that do not adversely affect each other. The active ingredients are suitably present in combination in amounts effective for the intended purpose.

The active ingredient can be incorporated into microcapsules prepared by, for example, coacervation techniques or interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), a glial drug delivery system (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences , 16th Ed., Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles, such as films or microcapsules.

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

G. Methods and compositions

Any of the anti-MCSP antibodies provided herein can be used in therapeutic methods.

In one aspect, an anti-MCSP antibody for use as a medicament is provided. In other aspects, an anti-MCSP antibody for use in treating cancer is provided. In certain embodiments, an anti-MCSP antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-MCSP antibody for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of an anti-MCSP antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent, such as described below. In other embodiments, the invention provides anti-MCSP antibodies for use in the treatment of melanoma. The "individual" of any of the above embodiments is preferably human.

In another aspect, the invention provides the use of an anti-MCSP antibody in the manufacture or preparation of a medicament. In one embodiment, the medical system is for treating cancer. In another embodiment, the medicament is for use in a method of treating cancer, the method comprising administering to the individual having the cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent, such as described below. The "individual" of any of the above embodiments may be human.

In another aspect, the invention provides methods for treating cancer. In one embodiment, the method comprises administering to the individual having the cancer an effective amount of an anti-MCSP antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent, as described below. The "individual" of any of the above embodiments may be human.

In one embodiment, the cancer in the above aspect exhibits MCSP on the surface of its constituent cells. In one embodiment, the cancer in the above aspect is selected from the group consisting of skin cancer (including melanoma and basal cell carcinoma), glioma (including nerve glue) Blastoma), bone cancer (eg osteosarcoma) and leukemia (including ALL and AML). In one embodiment, the cancer in the above aspect is melanoma.

In another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-MCSP antibodies provided herein for use in, for example, any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-MCSP antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-MCSP antibodies provided herein and at least one other therapeutic agent, for example as described below.

The antibodies of the invention may be used in therapy alone or in combination with other agents. For example, an antibody of the invention can be co-administered with at least one other therapeutic agent.

The above combination therapies encompasses combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and administered separately (in this case, other therapeutic agents and/or adjuvants may be administered) The antibody of the invention is administered before, simultaneously and/or after). The antibodies of the invention may also be used in combination with radiation therapy.

The antibodies of the invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and (if desired for topical treatment) intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection), depending on the length of administration. This article covers a variety of dosing regimens including, but not limited to, single or multiple doses, bolus injections, and pulse infusions at different time points.

The antibodies of the invention can be formulated, administered and administered in a manner consistent with good medical practice. In this context, considerations include the particular disease being treated The disease, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the drug, the method of administration, the timing of the administration, and other factors known to the medical practitioner. The antibody need not (but optionally) be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. The agents are generally administered in the same dosages and in the administration routes described herein, or from about 1% to about 99% of the dosages described herein, or in any of the dosages or routes which are empirically/clinically determined to be suitable.

For the prevention or treatment of a disease, the appropriate dose of the antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and duration of the disease, and the administration The anti-system is used for prophylactic or therapeutic purposes, prior therapy, clinical history of the patient and response to antibodies, and judgments of the attending physician. The antibody is suitable for administration to a patient once or via a series of treatments. The type and severity of the disease, whether administered by, for example, one or more separate administrations or by continuous infusion, from about 1 μg/kg to 15 mg/kg (eg, 0.1 mg/kg to 10) The mg/kg) antibody can be administered to the patient's initial candidate dose. Depending on the above factors, a typical daily dose may range from about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment usually continues until the desired inhibition of the symptoms of the disease occurs. An exemplary dose of one of the antibodies will range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to the patient. The dose can be The administration is intermittent, such as once a week or once every three weeks (eg, to allow the patient to receive from about 2 to about 20 or, for example, about 6 doses of the antibody). A higher loading dose can be administered at the beginning, followed by one or more lower doses. The progress of this therapy can be easily monitored by conventional techniques and analysis.

It will be appreciated that instead of or in addition to the anti-MCSP antibody, the immunoconjugate of the invention can be used to carry out any of the above methods of formulation or treatment.

H. Products

In another aspect of the invention, an article of manufacture comprising a material useful for treating, preventing, and/or diagnosing the above conditions is provided. The article comprises a container and a label or package insert located on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container holds the composition itself or a combination of the composition with another effective therapeutic, prophylactic and/or diagnosing condition, and may have a sterile access barrier (eg, the container may be an intravenous solution bag or vial having a stopper pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the selected condition. Additionally, the article of manufacture may comprise (a) a first container comprising a composition, wherein the composition comprises an antibody of the invention; and (b) a second container comprising the composition, wherein the composition comprises another cytotoxic agent or other treatment Agent. The article of this embodiment of the invention may additionally comprise a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article may additionally comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution And dextrose solution. It can be another It includes other materials that are needed from a commercial and user perspective, including other buffers, thinners, filters, needles, and syringes.

It will be appreciated that any of the above articles may comprise an immunoconjugate of the invention in place of or in addition to an anti-MCSP antibody.

Instance

The following are examples of the methods and compositions of the present invention. It should be understood that a number of other embodiments may be practiced in light of the general description provided above.

Example 1 - Production of anti-MCSP antibodies Immunization and fusion tumor production

Balb/c mice were immunized intraperitoneally 4 times every 4 weeks with a synthetic peptide corresponding to aa 2177-2221 (SVPE AARTEAGKPE SSTPTGEPGPMASSPEPAVA KGGFLSFLEAN (SEQ ID NO: 2)) in a human MCSP sequence coupled to KLH, followed by 4 intraperitoneal immunizations, followed by MCOP-expressing Colo38 cells (Giacomini P, Natali P, Ferrone SJ Immunol. July 1985; 135(1): 696-702) were immunized twice. The initial immune line was performed in CFA, followed by all enhancement lines in IFA.

Serum tests were performed for blood collection and half-maximal serum titers were determined using MCSP peptide aa2177-2221 coupled to biotin and coated onto streptavidin ELISA microtiter plates. Intravenous boosting was performed in mice with a half-maximum titer of 1:50,000. Intravenous boosting was performed using 20 μg of MCSP peptide and Colo38 cells on day 4 before fusion. Three days after the intravenous boost, spleen cells were harvested and fused with Ag8 myeloma cells.

Screening and fusion tumor characterization

Screening for MCSP-specific antibodies begins with the identification of binding to anti-antibody An antibody to MCSP-biotin peptide aa 2177-2221 (SEQ ID NO: 2) of a tropectin microtiter plate. The positive pure line that binds to the immobilized MCSP peptide was then amplified in serum-free medium (Hyclone ADCF-Mab-Thermo Scientific, Cat. No. SH30349.02).

Colo38 cells that naturally overexpress a large number of human MCSPs were subjected to FACS analysis for binding to the native form of MCSP. A prostate cancer cell line PC3 which does not exhibit a detectable amount of MCSP was used as a negative control. To further characterize the specificity of the primary antibodies, double immunocytochemical analysis of Colo38 cells was performed using commercially available anti-MCSP antibodies (Invitrogen, Cat. No. 41-2000, pure LHM2) and chimerism. The combination of antibodies (expressing human Fc) was double stained. As shown by immunofluorescence labeling, one antibody LC007 strongly stained surface MCSP in Colo38 cells but was negative on PC3 cells.

Example 2: Chimeric

mRNA was isolated and transformed into cDNA using a commercially available kit from a fusion cell line expressing the antibody line LC007. The heavy chain (SEQ ID NO: 39) and light chain (SEQ ID NO: 38) of the cDNA isolate were sequenced and each fragment was fused to the constant regions of human IgGl and kappa.

The signal peptide expression sequence from human immunoglobulin was used in HEK-EBNA cells and purified using conventional protein A and size exclusion chromatography (SEC).

The binding activity was determined by the following method. The target cells were detached from the culture flask with cell dissociation buffer, and counted and checked for viability. The cells were resuspended in PBS-0.1% BSA and adjusted to 1.111 x 10 ⁶ (live) cells/ml. 180 μl of this suspension was transferred to each well of a round bottom 96-well plate (200,000 cells/well), centrifuged at 400 g for 4 min, and resuspended. 20 μl of antibody dilution (from 10 μg/ml to 0.002 μg/ml) in PBS-0.1% BSA was added to each well. The sample was centrifuged at 400 g for 4 min and allowed to resuspend. A secondary antibody FITC-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG Fcg specific fragment (Jackson Immuno Research Lab, accession number 109-096-098) was added and the sample was centrifuged at 400 g for 4 min and It resuspends. Fluorescence is measured in a flow cytometer such as the FACS Canto II. The titration results are shown in Figures 1 and 2. The antibody 9.2.27 described in Morgan AC Jr, Galloway DR, Reisfeld RA. Hybridoma. 1981; 1(1): 27-36 (the light and heavy chain gene bank accession numbers are: GI: 20797193 and GI, respectively) : 20797189) as a reference (Figure 2). Human melanoma cell lines Colo38, A2058 and A375 were used. Giacomini et al. 1985 (for Colo38). Marquardt H, Todaro GJ. J Biol Chem. May 10, 1982; 257(9): 5220-5 (for A2058). Geiser M, Schultz D, Le Cardinal A, Voshol H, García-Echeverría C. Cancer Res. February 15, 1999; 59(4): 905-10 (for A375).

Example 3: Determination of binding epitopes of LC007 antibody on MCSP antigen

The LC007 antibody showed good binding on melanoma cells but only weakly bound on the original immunogen. Therefore, epitope mapping of antibody LC007 was performed to determine the exact binding site on the antigen. To this end, several truncated forms of MCSP antigen are produced, each containing a different number of membrane proximal repeat regions of human MCSP (referred to as CSPG repeats). Staub E., et al., FEBS Lett. 527: 114-118 (2002).

Construct 1 contains CSPG repeat 15 (SEQ ID NO: 4), construct 2 contains CSPG repeats 14-15 (SEQ ID NO: 5), and construct 3 contains CSPG repeats 13-15 (SEQ ID NO: 6) And construct 4 contains CSPG repeats 12-15 (SEQ ID NO: 7). Figure 3 provides a schematic representation of the structure of a MCSP containing a CSPG repeat. These constructs contain the original transmembrane region and are rendered on HEK-EBNA cells for detection of LC007 binding by FACS. Figure 4 shows the results of this experiment. Constructs comprising only the MCSP repeat 15 and the native transmembrane domain did not show any significant binding. In contrast, all of the constructs including domains 14 and 15 showed significant binding. This indicates that the binding epitope is located within the repeat sequence 14, or only when there is a repeat sequence 14 and may also include a portion of the repeat sequence 15 or an unstructured region between the CSPG repeat sequence and the transmembrane domain.

Example 4: Determination of cross-reactivity with human and cynomolgus antigens

Expression constructs were generated which included the C-terminal portion of the cynomolgus MCSP protein, the signal peptide for secretion, and the N-terminal FLAG-tag (SEQ ID NO: 8) to test for cross-reactivity to cynomolgus antigens. This domain is referred to as the D3 domain, Tillet, F. et al., J. Biol. Chem. 272: 10769-10776 (1997). A similar construct was produced against the human counterpart (SEQ ID NO: 9). The expression plasmid encoding the two constructs was electroporated into HEK-EBNA cells, and the expression was confirmed with an anti-FLAG antibody. The binding of the LC007 antibody was then tested by flow cytometry. Figure 5 shows that the affinity of the antibody LC007 in combination with the cynomolgus monkey construct is similar to that of the corresponding human expression construct. Sex.

Example 5: LC007 antibody modified by glycosylation

The glycosylation-modified variant of the LC007 antibody is produced by co-transfection of the antibody expression vector with the GnT-III glycosyltransferase expression vector or with the GnT-III expression vector plus the Golgi mannosidase II expression vector.

Example 6 ADCC of a glycosylation-modified LC007 antibody ADCC analysis

Humans at a 1:19 target:effector ratio were measured by fluorescence dye retention during the 16 h incubation period at 37 °C in the presence of different concentrations of glycosylated LC007 antibody and control antibody samples Lysis of lymphocytes (effectors) on Colo38 human malignant melanoma cells (target). Kolber et al., 1988, J. Immunol. Methods 108: 255-264. IMR-32 cells were labeled with the fluorescent dye calcein AM for 20 min (final concentration 3.3 μM). Labeled cells (80,000 cells/well) were incubated with different concentrations of glycosylated LC007 antibody and control antibody samples for 1 h. Then, a monocyte depleted mononuclear cell (1,500,000 cells/well) was added and the cell mixture was incubated at 37 ° C and 5% CO ₂ for 16 h. The supernatant was discarded and the cells were washed once with HBSS and lysed in Triton X-100 (0.1%). The retention of the fluorescent dye in Colo38 cells was measured with a fluorometer (Perkin Elmer, luminescence spectrometer LS 50B, Foster City, Calif.) and calculated relative to the total lysis control (obtained from target exposure to detergent rather than exposure) Specific cleavage of antibodies). The signal in the absence of antibody was set to 0% cytotoxicity. Each antibody concentration was analyzed in triplicate and the assay was repeated three times separately. As shown in Figure 6, the LC007 antibody (LC007 wt), which was not glycosylated, exhibited an ADCC effect. The glycosylated LC007 antibody (LC007 g2) showed enhanced ADCC compared to unmodified glycosylation LC007. Thus, the LC007 antibody, which has not been glycosylated, exhibits certain ADCC activity itself, which can be further enhanced by sugar modification. In contrast, the anti-MCSP antibody MHLG KV9 G2 (which is a humanized form of antibody 225.28S, such as Buraggi G et al, Int J Biol Markers. January-April 1986; 1(1): 47-54 None of the significant ADCC inductions shown in this analysis. The binding epitope for the 225.28 antibody is determined to be located within the N-terminal portion or distal portion of the membrane of the MCSP antigen. A glycosylated GA201 antibody that binds to the EGF receptor (which is not present on Colo38 cells) is included as a control. The absence of this antibody in ADCC indicates that activation of NK cells must be carried out via a target deposited on tumor cells.

Figure 7 shows that the ADCC of the glycosylated LC007 antibody was also observed for the human U86MG neutrophil blastoma cell line.

Example 7 Humanization of LC007 antibody modified by glycosylation

The humanized program follows the classical ring grafting procedure (Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Nature. May 29-June 4, 1986; 321 (6069): 522-5.P .Carter et al; Proc. Natl. Acad. Sci. USA; Vol. 89, pp. 4285-4289, May 1992). Briefly, the CDRs of the murine antibodies (SEQ ID NOS. 10, 11, 12, 14, 15 and 16) were grafted to the human framework sequence (IMGT accession numbers for the light chain are IGKV1D-39*01 and IGKJ1 and are heavy The chain's IMGT accession number is IGHV4-31*02 and IGHJ4), thus obtaining an amino acid containing The heavy chain of SEQ ID NO: 29 and the antibody comprising the light chain of the amino acid sequence SEQ ID NO: 28.

The antibody construct is optimized to retain binding affinity for the target MCSP antigen. Figure 8 shows the binding properties of different humanized variants. Substituting human residues Val71 and Arg94 for their corresponding murine counterparts (arginine and aspartate, respectively), as determined by antibody constructs with such human residues The combination. As shown in Figure 8, at position 94 (Kabat numbering) of the heavy chain, there is a construct Arg of M4-2 ML1 (SEQ ID NO: 30 (corresponding to D98R in this sequence)) and at position 74 of the heavy chain ( Kabat numbering) M4-6 ML1 with Val (SEQ ID NO: 33 (corresponding to R72V in this sequence)) showed reduced binding to the MCSP antigen, indicating that these residues are associated with binding specificity of the antibody. The constructs of the corresponding murine counterparts (arginine and aspartate) at each position retain binding activity, for example, they have M4-1 (SEQ ID NO: 29) and M4-3 ( An antibody to the heavy chain construct of SEQ ID NO: 32).

The CDR-H1 residue Asn35 is substituted with the corresponding human germline serine residue. As shown in Figure 8, the construct M4-7 ML1 (SEQ ID NO: 25) containing this substitution showed a decrease in binding to the target MCSP antigen, indicating that this residue is also involved in retaining antigen binding strength.

Other constructs indicate the correlation of other residues in the binding properties of the anti-MCSP antibodies. Substitution of the arginine residue with serine acid at position 7 of HVR-L1 (SEQ ID NO: 21) reduced the binding activity to the MCSP antigen. Substitution of aspartic acid for aspartic acid tyrosine at position 1 of HVR-L2 SEQ ID NO: 22 and replacement of alanine with threonine at position 2 also reduced binding activity to MCSP antigen.

Example 8 ADCC of a humanized variant of a glycosylation-modified LC007 antibody

The ADCC activity of the humanized variant of the glycosylated HP007 antibody was measured by lactate dehydrogenase using Colo38 cells as target cells. Human peripheral blood mononuclear cells (PBMC) were used as effector cells and were prepared using Histopaque-1077 (Sigma Diagnostics, St. Louis, Mo. 63178 USA) essentially following the manufacturer's instructions.

Briefly, venous blood was taken from healthy volunteers using a heparin syringe. The blood was diluted 1:0.75-1.3 with PBS (without Ca ⁺⁺ or Mg ⁺⁺ ) and layered on Histopaque-1077. The gradient was centrifuged at 400 x g for 30 min at room temperature (RT). The intermediate phase containing PBMC was collected and washed with PBS (50 ml PBS from each of the two gradients) and harvested by centrifugation at 300 x g for 10 minutes at RT. After the pellet was resuspended in PBS, PBMCs were counted and washed again by centrifugation at 200 x g for 10 minutes at RT. The cells are then resuspended in appropriate media for subsequent procedures.

For PBMC and NK cells, the effector:target ratios for ADCC analysis were 25:1 and 10:1, respectively. Effector cells were prepared in AIM-V medium at appropriate concentrations for addition in 50 μl/well in round bottom 96-well plates. The target cell line is Colo30 cells. The target cells were washed in PBS, counted, and resuspended in AIM-V at 0.3 × 10 ⁶ /ml to add 30,000 cells / 100 μl / microwell. The antibody was diluted in AIM-V, added to pre-plated target cells at 50 μl and allowed to bind to the target for 10 minutes at RT. Effector cells were then added and the plates were incubated for 4 hours at 37 ° C in a humidified atmosphere containing 5% CO ₂ . Target cell killing was assessed using a cytotoxicity test kit (Roche Diagnostics, Rotkreuz, Switzerland) by measuring lactate dehydrogenase (LDH) released from damaged cells. After 4 hours of incubation, the plates were centrifuged at 800 x g. Transfer 100 μl of supernatant from each well to a new clear flat-bottom 96-well plate. Add 100 μl of colored matrix buffer to each well from the kit. The Vmax value of the color reaction was determined using an SOFTmax PRO software (Molecular Devices, Sunnyvale, Calif. 94089, USA) at 490 nm for at least 10 min in an ELISA reader. Spontaneous LDH release was measured from wells containing only target and effector cells but no antibodies. Maximum release was determined from wells containing only target cells and 1% Triton X-100. The percentage of antibody-mediated specific killing was calculated as follows: ((x-SR)/(MR-SR)*100, where the mean of Vmax at x for specific antibody concentrations and the mean Vmax for SR spontaneous release And the average value of the maximum release Vmax of the MR system.

Figure 9 shows the results of this analysis and confirms that the humanized variant retains the ADCC activity of the glycosylated parental LC007 antibody.

After washing with 5 mM borate buffer containing 0.1% Triton X-100 and cell lysis, viable target cells were further quantified by calcein measurement using the assay as described in Example 6 (Wallac Victor 3 1420 Multilabel Counter). The results of this analysis are shown in Figure 10.

Example 9 Analysis of mouse xenografts 9.1 MV3 cells in FcgR3 transgenic SCID mice

20 FcgR3A tg SCID mice (purchased from Charles River, Lyon, France) were maintained in IVC with a daily cycle of 12 h light/12 h dark according to the agreed guidelines (GV-Solas; Felasa; TierschG) Under the condition of gas cage). Review and approve experimental research programs by local governments (P 2005086). Animals were maintained for one week after arrival to accommodate the new environment and to observe. Continue regular health monitoring.

The MV3 tumor cell line (van Muijen GN et al., Int J Cancer. 48(1): 85-91 (1991)) was cultured in a conventional manner at 37 ° C in a water saturated atmosphere of 5% CO ₂ . 10% fetal calf serum (Invitrogen, Switzerland) in DMEM medium (GIBCO, Switzerland). The culture was passaged with trypsin/EDTA 1× (GIBCO, Switzerland) and separated every three days. On the day of injection, tumor cells were harvested from a culture flask (Greiner Bio-One) using trypsin-EDTA (Gibco, Switzerland) and transferred to 50 ml of medium, washed once in AIM V (Gibco, Switzerland) and allowed to It resuspends. After washing again with AIM V, the cell concentration was determined using a cell counter. 0.2 x 10 ⁶ cells in 200 μl of Aim V medium were injected into the tail vein of each FcgR3A tg SCID mouse.

treatment

Xenograft mice were assigned to treatment or vehicle control groups, each consisting of nine mice. A 25 mg/kg glycosylated humanized anti-MCSP mAb M4-3 ML2 was administered intravenously to the treatment group. The vehicle was administered intravenously only to the vehicle control group. Both groups received three doses on days 7, 14, and 21.

Statistical analysis was performed on data obtained from treatment using the Mantel-Cox test (p=0.0033) and the Gehan-Breslow-Wilcoxon test (p=0.0039).

result

As shown in Figure 11, in this model, glycosylated humanized anti-MCSP antibodies significantly prolonged survival compared to vehicle controls.

9.2 MDA-MB-435 cells in FcgR3 transgenic SCID mice

MDA-MB435 cells were originally obtained from ATCC and were deposited in the Glycart internal cell bank after amplification. The MDA-MB435 tumor cell line was cultured in a conventional manner at 37 ° C in a water saturated atmosphere of 5% CO ₂ in RPMI medium supplemented with 10% fetal calf serum (Invitrogen, Switzerland) and 1% Glutamax (GIBCO, Switzerland). in. The culture was passaged with trypsin/EDTA 1× (GIBCO, Switzerland) and separated every three days.

FcgR3A tg SCID mice (purchased from Charles River, Lyon, France) were maintained in IVC with a daily cycle of 12 h light/12 h dark according to the agreed guidelines (GV-Solas; Felasa; TierschG) (Separation of ventilation cages) Under the conditions). The experimental research program was reviewed and approved by the local government (P 2005086). Animals were maintained for one week after arrival to accommodate the new environment and to observe. Continue regular health monitoring.

On the day of injection, tumor cells were harvested from a culture flask (Greiner Bio-One) using trypsin-EDTA (Gibco, Switzerland) and transferred to 50 ml of medium, washed once in AIM V (Gibco, Switzerland) and allowed to It resuspends. After washing again with AIM V, the cell concentration was determined using a cell counter. 0.2 x 10 ⁶ cells in 200 μl of Aim V medium were injected into the tail vein of each FcgR3A tg SCID mouse.

treatment

Xenograft mice were assigned to a treatment or vehicle control group. Treatment The group was administered intravenously with a 25 mg/kg glycosylated chimeric anti-MCSP mAb. The vehicle was administered intravenously only to the vehicle control group. Both groups received three doses on days 7, 14, and 21.

result

As shown in Figure 12, in this model, glycosylated modified chimeric anti-MCSP antibodies significantly prolonged survival compared to vehicle controls.

9.3 MDA-MB-435 cells in FcgR3 transgenic SCID mice

The same protocol as in Example 9.2 was followed, but the humanized antibody M4-3 ML2 (containing VH of SEQ ID NO: 32 and VL of SEQ ID NO: 31) was compared to its parental chimeric antibody LC007. Both antibodies are glycosylated.

result

As shown in Figure 13, in this model, both the parental chimeric antibody LC007 and its glycosylated engineered variants significantly prolonged survival compared to vehicle controls.

The present invention has been described in some detail by way of illustration and example, and is not to be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

Figure 1 is a graph showing the results of FACs analysis showing the binding affinity of chimeric antibody LC007 to surface MCSP in Colo38 cells.

Figure 2 is a graph showing the results of FACs analysis showing the binding affinity of chimeric antibody LC007 to surface MCSP in A2058 and A375 cancer cells.

Figure 3 is a schematic representation of the structure of a MCSP containing a CSPG repeat.

Figure 4 is a graph showing the binding specificity of LC007 for MCSP CSPG repeat constructs.

Figure 5 is a graph showing the results of FACs analysis showing that the affinity of antibody LC007 in combination with a cynomolgus monkey construct is similar to the affinity of a corresponding human expression construct.

Figure 6 shows that it has not been glycosylated and modified by glycosylation. A plot of the ADCC effect of both LC007 antibodies.

Figure 7 is a graph showing the ADCC effect of a glycosylated LC007 antibody observed in a human U86MG neutrophil blastoma cell line.

Figure 8 is a graph showing the binding properties of several humanized variants of the LC007 antibody.

Figure 9 is a graph showing the ADCC activity of a humanized variant of LC007 retaining the glycosylated parental LC007 antibody.

Figure 10 is a graph showing the ADCC activity of a humanized variant of LC007 retaining the glycosylated modified parental LC007 antibody.

Figure 11 depicts survival curves showing that glycosylated engineered humanized anti-MCSP antibodies significantly prolonged survival in vehicle FcgR3A transgenic SCID mice with MV3 tumor cell lines compared to vehicle controls.

Figure 12 depicts survival curves showing that glycosylated engineered chimeric anti-MCSP antibodies significantly prolonged survival in vehicle FcgR3A transgenic SCID mice with MDA-MB-435 tumor cell lines compared to vehicle controls.

Figure 13 depicts survival curves showing both glycosylated modified chimeric anti-MCSP antibodies and their humanized variant M4-3 ML2 in FcgR3A transgenic SCID mice with MDA-MB-435 tumor cell lines The survival time was significantly prolonged compared with the vehicle control.

<110> Swiss company Rozcyliya

<120> Anti-MCSP Antibody

<130> 30603

<140> 101130535

<141> 2012/08/22

<150> EP 11178393.2

<151> 2011-08-23

<160> 44

<170> PatentIn version 3.5

<210> 1

<211> 2322

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 45

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 107

<212> PRT

<213> Homo sapiens

<400> 3

<210> 4

<211> 203

<212> PRT

<213> Homo sapiens

<220>

<221> DOMAIN

<222> (182)..(203)

<223> Transmembrane domain

<400> 4

<210> 5

<211> 310

<212> PRT

<213> Homo sapiens

<220>

<221> Domain

<222> (289)..(310)

<223> Transmembrane domain

<400> 5

<210> 6

<211> 419

<212> PRT

<213> Homo sapiens

<220>

<221> Domain

<222> (398)..(419)

<223> Transmembrane domain

<400> 6

<210> 7

<211> 545

<212> PRT

<213> Homo sapiens

<220>

<221> Domain

<222> (524)..(545)

<223> Transmembrane domain

<400> 7

<210> 8

<211> 643

<212> PRT

<213> Cynomolgus monkey

<400> 8

<210> 9

<211> 643

<212> PRT

<213> Homo sapiens

<400> 9

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 10

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 11

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L3

<400> 12

<210> 13

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 13

<210> 14

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 14

<210> 15

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 15

<210> 16

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H3

<400> 16

<210> 17

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 17

<210> 18

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H2

<400> 18

<210> 19

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 19

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 20

<210> 21

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L1

<400> 21

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 22

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 23

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-L2

<400> 24

<210> 25

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> HVR-H1

<400> 25

<210> 26

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Chimeric Antibody VL

<400> 26

<210> 27

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Chimeric Antibody VH

<400> 27

<210> 28

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody ML1 VL

<400> 28

<210> 29

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody M4-1 VH

<400> 29

<210> 30

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody M4-2 VH

<400> 30

<210> 31

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody ML2 VL

<400> 31

<210> 32

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody M4-3 VH

<400> 32

<210> 33

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody M4-6 VH

<400> 33

<210> 34

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Chimeric Antibody Light Chain

<400> 34

<210> 35

<211> 442

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Chimeric Antibody Heavy Chain

<400> 35

<210> 36

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody ML2 Light Chain

<400> 36

<210> 37

<211> 442

<212> PRT

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody M4-3 Heavy Chain

<400> 37

<210> 38

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> LC007 murine antibody light chain

<400> 38

<210> 39

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> LC007 murine antibody heavy chain

<400> 39

<210> 40

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> LC007 Chimeric Antibody Light Chain

<400> 40

<210> 41

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> LC007 Chimeric Antibody Heavy Chain

<400> 41

<210> 42

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody ML2 Light Chain

<400> 42

<210> 43

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> LC007 Humanized Antibody M4-3 Heavy Chain

<400> 43

<210> 44

<211> 21

<212> PRT

<213> Homo sapiens

<400> 44

Claims (32)

An isolated antibody that binds to a membrane proximal epitope of a human MCSP comprising a domain comprising a CSPG repeat, wherein the antibody has been glycosylated to modify an oligosaccharide in the Fc region, and wherein the antibody has a ratio of Glycosylation-modified antibody-enhanced ADCC effector function. The antibody of claim 1, wherein the domain comprising the CSPG repeat comprises CSPG repeat 14 (SEQ ID NO: 3). The antibody of claim 1 or 2, wherein the Fc region has a reduced number of fucose residues than the antibody that has not been glycosylated. The antibody of claim 1 or 2, wherein the antibody has a ratio of GlcNAc residues to fucose residues in the Fc region that is greater than the antibody that has not been glycosylated. The antibody of claim 1 or 2, wherein the Fc region has a ratio of dichomeric oligosaccharides that is increased by the antibody that has not been glycosylated. An antibody according to claim 1 or 2, wherein the anti-system monoclonal antibody. The antibody of claim 1 or 2, wherein the anti-system is a human, humanized or chimeric antibody. The antibody of claim 7, wherein the anti-systemic full length IgG class antibody. The antibody of claim 1 or 2, wherein the antibody comprises (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 14; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 15. And (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16. The antibody of claim 9, which further comprises (a) HVR-L1 comprising an amino acid sequence of SEQ ID NO: 10; (b) HVR-L2 comprising an amino acid sequence SEQ ID NO: 11; and (c) HVR-L3, which comprises the amino acid sequence SEQ ID NO: 12. The antibody of claim 1 or 2, wherein the antibody comprises (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 10; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO And 11(c)HVR-L3, which comprises the amino acid sequence SEQ ID NO: 12. The antibody of claim 1 or 2, wherein the antibody comprises (a) HVR-H1 comprising the amino acid sequence SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence SEQ ID NO: 18 And (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 16. The antibody of claim 12, which further comprises (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO: 11; (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 12. The antibody of claim 1 or 2, wherein the antibody comprises (a) HVR-L1 comprising the amino acid sequence SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence SEQ ID NO And 11(c)HVR-L3, which comprises the amino acid sequence SEQ ID NO: 12. The antibody of claim 1 or 2, which comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 27; (b) having at least the amino acid sequence SEQ ID NO: 26 a VL sequence of 95% sequence identity; or (c) a VH sequence as in (a) and a VL sequence as in (b). The antibody of claim 15, which comprises the VH sequence of SEQ ID NO:27. The antibody of claim 15, which comprises the VL sequence of SEQ ID NO: 26. The antibody of claim 15, which comprises the VH sequence of SEQ ID NO: 27 and the VL sequence of SEQ ID NO: 26. The antibody of claim 1 or 2 which comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 32; (b) having at least an amino acid sequence of SEQ ID NO: 31 a VL sequence of 95% sequence identity; or (c) a VH sequence as in (a) and a VL sequence as in (b). The antibody of claim 19, which comprises the VH sequence of SEQ ID NO:32. The antibody of claim 19, which comprises the VL sequence of SEQ ID NO:31. The antibody of claim 19, which comprises the VH sequence of SEQ ID NO: 32 and the VL sequence of SEQ ID NO: 31. An isolated nucleic acid encoding the antibody of any one of claims 1 to 22. A host cell comprising the nucleic acid of claim 23. A method of producing an antibody comprising culturing a host cell as claimed in claim 24 to produce the antibody. An immunoconjugate comprising the antibody of any one of claims 1 to 22 and a cytotoxic agent. A pharmaceutical formulation comprising the antibody of any one of claims 1 to 22 and a pharmaceutically acceptable carrier. An antibody according to claim 1 or 2 or an immunoconjugate of claim 26 for use as a medicament. Use of an antibody according to any one of claims 1 to 22, or an immunoconjugate of claim 26, for the manufacture of a medicament for the treatment of cancer. The use of claim 29, wherein the cancer is a cancer that exhibits MCSP. The use of claim 30, wherein the cancer is selected from the group consisting of skin cancer (including melanoma and basal cell carcinoma), glioma (including neutrophil blastoma), bone cancer (eg osteosarcoma), and Leukemia (including ALL and AML). Use of an antibody according to any one of claims 1 to 22 for the manufacture of a medicament for inducing cell lysis.