Brain-specific angiogenesis inhibitor 1 (BAI1) antibody and uses thereof

Abstract

The present disclosure provides antibodies that bind to the human adhesion G protein-coupled receptor B1 (BAI1) protein, compositions containing the antibodies, and their use in detection, diagnostic, and therapeutic methods. The disclosure also provides sequences of the complementarity-determining regions (CDRs) of the variable heavy chain (VH) and variable left chain (VL), as well as the amino acid sequences of the VH and VL.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as a text file entitled 18909900502SEQ, 25 kilobytes in size, created on November 2, 2020. That Sequence Listing is incorporated herein by reference.

[0002] The present disclosure is directed to antibodies and fragments thereof that bind to human brain-specific angiogenesis inhibitor 1 (BAI1) (also known as adhesion G protein-coupled receptor B1 (ADGRB1)) protein, compositions comprising the antibodies and fragments thereof, and uses of the antibodies and fragments thereof in detection, cell isolation, depletion, diagnostic, and therapeutic methods. [Background technology]

[0003] BAI1 is expressed in cells of the monocyte lineage, astrocytes, neurons, and Myo / Nog cells, which express the skeletal muscle-specific transcription factor MyoD and the bone formation inhibitor Noggin. + BAI1 cells are involved in innate immunity, wound healing, embryonic development and neuroprotection. + The wound healing properties of the cell reflect, in part, the molecule's role in phagocytosis of apoptotic cells and Gram-negative bacteria via its binding to phosphatidylserine. + Another function of a subpopulation of cells is to differentiate into myofibroblasts. While myofibroblast contraction can promote wound closure, it can also disrupt tissue morphology and, therefore, function. For example, secondary cataract or posterior capsule opacification (PCO) is a vision-impairing lens disease that occurs in some adults and most children after cataract surgery. BAI1 in the lens + The cells become myofibroblasts, create folds within the capsule, and form a thick basement membrane around the lens. Deformations in the lens capsule affect vision.

[0004] In addition to its role in phagocytosis, BAI1 is metabolized to produce an inhibitor of angiogenesis (the process by which blood vessels form from pre-existing blood vessels). In the retina, angiogenesis is a sight-threatening factor in diabetic retinopathy and wet macular degeneration. Angiogenesis is also essential for tumor growth; tumors cannot grow larger than a few millimeters without additional angiogenesis. BAI1 + BAI1 cells are present in the retina and tumors. In some cancers, BAI1 is downregulated as the disease progresses, thereby favoring angiogenesis. However, angiogenesis is also important for the distribution of chemotherapeutic agents, such as those attached to viruses. Summary of the Invention

[0005] The present disclosure provides an antibody or antigen-binding fragment thereof that binds to human BAI1 protein, comprising or consisting of the amino acid sequence set forth in SEQ ID NO:7, and a variable-heavy (V H The first complementarity-determining region (CDR) in the V H - CDR1) and the amino acid sequence set forth in SEQ ID NO: 8 or consisting of the amino acid sequence thereof; H The second CDR in the V H -CDR2) and the amino acid sequence set forth in SEQ ID NO: 9 or consisting of the amino acid sequence thereof; H The third CDR in the V H -CDR3) and a variable light chain (V) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10. L ) chain, L - CDR1) and the amino acid sequence set forth in SEQ ID NO: 11, or consisting of the amino acid sequence thereof; L The second CDR in the V L -CDR2) and the amino acid sequence set forth in SEQ ID NO: 12, or consisting of the amino acid sequence thereof; L The third CDR in the V L -CDR3).

[0006] The disclosure also provides an isolated nucleic acid molecule encoding the heavy chain of the antibody or antigen-binding fragment thereof, wherein the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:2 or SEQ ID NO:3.

[0007] The disclosure also provides an isolated nucleic acid molecule encoding the light chain of the antibody or antigen-binding fragment thereof, wherein the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0008] The present disclosure also provides a method for detecting BAI1-expressing cells, comprising contacting the cells with an antibody or antigen-binding fragment thereof described herein and detecting the antibody or antigen-binding fragment thereof. The detection can be performed in fixed tissues, cell cultures, and live animals for cell tracking and bioimaging.

[0009] The present disclosure also provides methods for purifying BAI1, comprising contacting the molecule with an antibody or antigen-binding fragment thereof described herein. These methods include, for example, immunoprecipitation and column purification.

[0010] The present disclosure also provides methods for isolating live cells expressing BAI1, comprising contacting the cells with an antibody or antigen-binding fragment thereof of the present invention and separating cells bound by the antibody or antigen-binding fragment described herein from unbound cells. These methods include, for example, fluorescence-activated cell sorting (FACS) and magnetic cell sorting.

[0011] The present disclosure also provides methods for analyzing BAI1 function, including contacting molecules and live cells in vitro and in vivo with the antibodies or antigen-binding fragments thereof described herein, including, for example, blocking receptors and fragments thereof in enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation, Western blotting, and live cell assays.

[0012] The present disclosure also provides methods for killing BAI1-expressing cells in vitro, in animals to determine cell function, and in human patients in need thereof, comprising administering an antibody or antigen-binding fragment thereof described herein.

[0013] The present disclosure also provides methods for treating BAI1-expressing cancers in cell culture, tumor-bearing animals, and tumor-bearing humans, comprising administering an antibody or antigen-binding fragment thereof described herein.

[0014] The present disclosure also provides methods for treating posterior capsule opacification (PCO), comprising administering to tissue culture, animals, and human patients in need thereof an antibody or antigen-binding fragment thereof described herein.

[0015] The present disclosure also provides methods for treating fibrosis, comprising administering to cultured cells, animals, and human patients in need thereof an antibody or antigen-binding fragment thereof described herein.

[0016] The present disclosure provides a method for promoting wound healing, comprising administering to a human patient in need thereof any of the antibodies or antigen-binding fragments thereof described herein, or BAI1. + Also provided are methods that include administering cells to the wound. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows the antigen identification workflow using the membrane proteome array (MPA) platform. [Figure 2]A shows the optimization of immunodetection of G8 lot 3 / 21 / 13 in HEK-293T cells. Each point represents the average of four replicates. The recommended working concentration for screening of each antibody, based on calculated signal-to-background (S / B) and raw signal values, is highlighted in red. B shows the optimization of immunodetection of G8 lot PS30170160 in HEK-293T cells. Each point represents the average of four replicates. The recommended working concentration for screening of each antibody, based on calculated signal-to-background (S / B) and raw signal values, is highlighted in red. [Figure 3A] Figure 1 shows the identification of membrane protein binding targets in G8 lot 3 / 21 / 13. Nonspecific fluorescence was considered any value not exceeding 3 standard deviations above noise (dashed line). Targets that showed increased antibody binding are displayed above the dashed line and are colored red. [Figure 3B] Figure 1 shows the identification of membrane protein binding targets in G8 lot PS30170160. Nonspecific fluorescence was considered any value not exceeding 3 standard deviations above noise (dashed line). Targets that showed increased antibody binding are displayed above the dashed line and are colored red. [Figure 4] A shows the binding validation results for G8 lot 3 / 21 / 13. Each point represents the average of four replicates. B shows the binding validation results for G8 lot PS30170160. Each point represents the average of four replicates. [Figure 5] 1 shows the results of identification of the heavy and light chains of mouse G8 IgM by RACE. [Figure 6] Shown are the results of ELISA assays for G8 IgM (boxes) and anti-BAI1 IgG (circles, also referred to herein as "G8 IgG").

[0018] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments as claimed herein. DETAILED DESCRIPTION OF THE INVENTION

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Throughout the specification and claims, various terms relating to aspects of the present disclosure are used. Such terms shall have their ordinary meaning in the art unless otherwise indicated. Other terms that are specifically defined shall be interpreted consistent with the definitions set forth herein.

[0020] Unless expressly stated otherwise, in no event is it intended that any method or embodiment defined herein be construed as requiring its steps to be performed in any particular order. Unless a method claim specifically recites its steps as being limited to a particular order, no order is intended to be specified in any claim or description in any respect. This applies regardless of any possible implicit standard of interpretation, including matters of logic regarding the ordering of steps or workflow, the plain meaning derived from grammatical structure or punctuation, or the number or type of embodiments described herein.

[0021] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] As used herein, the term "about" means that the recited numerical value is approximate and that small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless the context indicates otherwise, "about" means that the numerical value can vary by ±10% and still fall within the range of the disclosed embodiments.

[0023] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms thereof, including, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies).

[0024] As used herein, the phrase "antigen-binding fragment thereof" refers to an antibody fragment capable of binding to an antigen, including, but not limited to, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a scFv fragment, a scFv-Fc fragment, a diabody fragment, a bispecific diabody fragment, a trispecific triabody fragment, a minibody fragment, a monospecific Fab2 fragment, a bispecific Fab2 fragment, a trispecific Fab3 fragment, a nanobody fragment, an IgNAR fragment, a V-NAR fragment, a hcIgG fragment, and a VhH fragment.

[0025] As used herein, the phrase "chimeric antibody" refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically a constant region selected from a human immunoglobulin template.

[0026] As used herein, the term "Fv" fragment refers to the minimum antibody fragment that contains an intact target recognition and binding site. This region consists of a non-covalently associated dimer of one heavy-chain variable domain and one light-chain variable domain (V H -V L In this configuration, the three CDRs of each variable domain interact to form the V H -V L The target binding site is formed on the surface of the dimer.

[0027] As used herein, the phrase "human antibody" refers to an antibody having the amino acid sequence of a human immunoglobulin, and includes antibodies isolated from a human immunoglobulin library or from an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins.

[0028] As used herein, the phrase "humanized antibody" includes chimeric antibodies or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of its CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of its framework (FR) regions being those of a human immunoglobulin sequence. A humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence.

[0029] As used herein, the phrase "in need of" means that the "subject" or "patient" has been identified as being in need of a particular method, prevention, or treatment. In some embodiments, the identification can be by any diagnostic means. The "subject" or "patient" can be in need of any of the methods, preventions, and treatments described herein.

[0030] As used herein, a "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, which may include DNA and / or RNA, and which may be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid can also be referred to as its complement.

[0031] As used herein, the phrase "primatized antibody" refers to an antibody comprising monkey variable regions and human constant regions.

[0032] As used herein, the phrase "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (such as polyadenylation signals) that control the transcription or translation of the antibody chain genes.

[0033] As used herein, the term "single-chain Fv" or "scFv" refers to the variable domains of the heavy and light chains (V H Domain and V L A single-chain Fv antibody refers to a single-chain Fv antibody in which multiple scFv domains (scFv domains) are linked together to form a single chain. Generally, the Fv polypeptide further comprises a polypeptide linker between its V domains that enables the scFv to form the desired structure upon target binding. H Domains and V L Include between domains.

[0034] As used herein, the terms "subject" and "patient" are used interchangeably. A subject includes any animal, including mammals. Mammals include, but are not limited to, livestock (e.g., horses, cows, pigs), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates (e.g., monkeys). In some embodiments, the subject or patient is human.

[0035] As used herein, "V H " refers to the variable region of an immunoglobulin heavy chain of an antibody, including, for example, the heavy chain of an Fv fragment, scFv fragment or Fab fragment.

[0036] As used herein, "V L " refers to the variable region of an immunoglobulin light chain of an antibody, including the light chain of, for example, an Fv, scFv, dsFv or Fab fragment.

[0037]

[0038] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to an epitope in the human BAI1 protein that comprises or consists of the following amino acids: Y83, F135, W415, W418, L420, T424, R432, R434, I458, W473, W476, R545, Y639, and L776. In some embodiments, the epitope in the human BAI1 protein to which the antibodies or antigen-binding fragments thereof described herein bind comprises or consists of at least 4-14, at least 4-13, at least 4-12, at least 4-11, at least 4-10, at least 4-9, at least 4-8, at least 4-7, or at least 4-6 of the above amino acids. In some embodiments, the epitope on the human BAI1 protein to which the antibodies or antigen-binding fragments thereof described herein bind comprises or consists of at least 6-14, at least 6-13, at least 6-12, at least 6-11, at least 6-10, at least 6-9, or at least 6-8 of the above amino acids. In some embodiments, the epitope on the human BAI1 protein to which the antibodies or antigen-binding fragments thereof described herein bind comprises or consists of at least 7-14, at least 7-13, at least 7-12, at least 7-11, at least 7-10, or at least 7-9 of the above amino acids. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to an epitope on the human BAI1 protein that comprises or consists of the following amino acids: F135, W415, W418, L420, T424, R432, R434, and I458. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to an epitope in the human BAI1 protein that includes or consists of the following amino acids: W415, W418, L420, T424, R432, R434, and I458.In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to an epitope in the human BAI1 protein that includes or consists of the following amino acids: T424, R432, R434, and I458.

[0039] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the human BAI1 protein. In some embodiments, the antibody or antigen-binding fragment thereof comprises three V H CDR and three Vs L Contains CDRs.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence GYSITSDY (SEQ ID NO: 7). H The first CDR in the V H In some embodiments, this V H - CDR1 may comprise or consist of one, two or three conservative amino acid substitutions.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence of SYSGS (SEQ ID NO: 8). H The second CDR in the V H In some embodiments, this V H - CDR2 may comprise or consist of one or two conservative amino acid substitutions.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence AQGYAMDY (SEQ ID NO: 9). H The third CDR in the V H In some embodiments, this V H- CDR3 may comprise or consist of one, two or three conservative amino acid substitutions.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises a V L The first CDR in the V L In some embodiments, this V L - CDR1 may comprise or consist of 1, 2, 3 or 4 conservative amino acid substitutions.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence YASQSIS (SEQ ID NO: 11). L The second CDR in the V L In some embodiments, this V L - CDR2 may comprise or consist of one or two conservative amino acid substitutions.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence QNGHSFPFT (SEQ ID NO: 12). L The third CDR in the V L In some embodiments, this V L - CDR3 may comprise or consist of one, two or three conservative amino acid substitutions.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence GYSITSDY (SEQ ID NO: 7). H The first CDR in the VH -CDR1) and the amino acid sequence SYSGS (SEQ ID NO: 8) or consisting of the amino acid sequence H The second CDR in the V H -CDR2) and the amino acid sequence AQGYAMDY (SEQ ID NO: 9) or consisting of the amino acid sequence H The third CDR in the V H -CDR3) and the amino acid sequence RASQSISDYLH (SEQ ID NO: 10) or consisting of the amino acid sequence L The first CDR in the V L -CDR1) and an amino acid sequence called YASQSIS (SEQ ID NO: 11) or consisting of the amino acid sequence L The second CDR in the V L -CDR2) and the amino acid sequence QNGHSFPFT (SEQ ID NO: 12) or consisting of the amino acid sequence L The third CDR in the V L -CDR3). In some embodiments, the CDRs may have conservative amino acid substitutions as described herein.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence SDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCANAQGYAMDYWGQGTSVTVSS (SEQ ID NO: 1). H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain consisting of the amino acid sequence set forth in SEQ ID NO:1. H Contains chains.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof has a V or VL sequence that comprises or consists of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO:1. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO:1. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO:1. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO:1. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO:1. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO:1. H In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO:1. H In some embodiments, the V H The chain may be any of the V H In some embodiments, the V H The CDRs comprise the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In some embodiments, the V HThe chain comprises or consists of 1, 2, 3, 4, 5, 6, 7, or 8 conservative amino acid substitutions. H The chains may comprise or consist of 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitutions in addition to the CDRs.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of the amino acid sequence DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK (SEQ ID NO: 4). L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain consisting of the amino acid sequence set forth in SEQ ID NO:4. L Contains chains.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises or consists of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO:4. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO:4. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO:4. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO:4. LIn some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO:4. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO:4. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO:4. L In some embodiments, the V L The chain may be any of the V L In some embodiments, the V L The CDRs comprise the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, the V L The chain contains 1, 2, 3, 4, 5, 6, 7, or 8 conservative amino acid substitutions. L The chains may contain 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitutions outside of the CDRs.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof comprises a V H a V chain comprising the amino acid sequence set forth in SEQ ID NO:4; L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain consisting of the amino acid sequence set forth in SEQ ID NO:1. H a V chain consisting of the amino acid sequence set forth in SEQ ID NO: 4; L Contains chains.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof has a V or VL sequence that comprises or consists of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO:1. H and a V chain comprising or consisting of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity with a SEQ ID NO. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO:1. H and a V chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity with SEQ ID NO:4. L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO:1. H a V chain comprising or consisting of an amino acid sequence that has at least about 90% sequence identity with SEQ ID NO:4; L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO:1. H a V chain comprising or consisting of an amino acid sequence that has at least about 92% sequence identity with SEQ ID NO:4; L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO:1. H and a V chain comprising or consisting of an amino acid sequence that has at least about 94% sequence identity with SEQ ID NO:4. LIn some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO:1. H a V chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity with SEQ ID NO:4; L In some embodiments, the antibody or antigen-binding fragment thereof comprises a V chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO:1. H a V chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity with SEQ ID NO:4; L In some embodiments, the V H Chain and V L The chain may be any of the V H CDR and V L In some embodiments, the V H The CDRs comprise the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In some embodiments, the V L The CDRs comprise or consist of the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, the V H Chain and V L Each chain can contain or consist of 1, 2, 3, 4, 5, 6, 7, or 8 conservative amino acid substitutions. H Chain and V L Each chain can comprise or consist of 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitutions in addition to the CDRs.

[0053] The antibody or antigen-binding fragment thereof can be of any isotype. In some embodiments, the antibody is an IgM antibody or an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the IgG1 antibody is an IgG1Glm1, IgG1Glm2, IgG1Glm3, IgG1Glm17, IgG1nGlmI, IgG1nGlm2, or IgG1nGlm17 allotype antibody. In some embodiments, the antibody is an IgG1Glm17 allotype antibody.

[0054] In some embodiments, the variant IgG1 heavy chain is paired with a κ light chain of the allotype Km1, Km2, or Km3. In some embodiments, the variant IgG1 heavy chain is paired with a λ light chain.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof is humanized and is an IgG. In some embodiments, the antibody or antigen-binding fragment thereof is humanized and is an IgG1.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof. Methods for producing chimeric antibodies are known in the art (see Morrison, Science, 1985, 229, 1202-1207; Oi et al., BioTechniques, 1986, 4, 214-221; Gillies et al., J. Immunol. Methods, 1985, 125, 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397).

[0057] In some embodiments, the chimeric antibody or antigen-binding fragment thereof is a primatized chimeric antibody or antigen-binding fragment thereof. Methods for making primatized antibodies are known in the art (see U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780).

[0058] In some embodiments, the chimeric antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. Methods for making humanized antibodies are known in the art (Riechmann et al., Nature, 1988, 332, 323-327; Padlan, Mol. Immunol., 1991, 28, 489-498; Studnicka et al., Prot. Eng., 1994, 7, 805-814; Roguska et al., J. Immunol., 1994, 7, 805-814). (See, e.g., U.S. Pat. Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370, and 5,565,332).

[0059] In some embodiments, the humanized antibody or antigen-binding fragment thereof is VQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCANAQGYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13.

[0060] In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO: 13. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO: 13. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO: 13. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO: 13. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO: 13. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO: 13. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 13. In some embodiments, the heavy chain comprises a V H In some embodiments, the heavy chain CDRs comprise any of the CDRs set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In some embodiments, the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions in addition to the CDRs.

[0061] In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of the amino acid sequence DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14). In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0062] In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 14. In some embodiments, the light chain comprises a V LIn some embodiments, the light chain CDRs comprise any of the CDRs set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, the light chain comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the light chain comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions in addition to the CDRs.

[0063] In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO: 13, and a light chain comprising or consisting of an amino acid sequence that has at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, or at least about 98% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO: 13, and a light chain comprising or consisting of an amino acid sequence that has at least about 85% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain that comprises or consists of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO: 13, and a light chain that comprises or consists of an amino acid sequence that has at least about 90% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain that comprises or consists of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO: 13, and a light chain that comprises or consists of an amino acid sequence that has at least about 92% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain that comprises or consists of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO: 13, and a light chain that comprises or consists of an amino acid sequence that has at least about 94% sequence identity to SEQ ID NO: 14. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO: 13, and a light chain comprising or consisting of an amino acid sequence that has at least about 96% sequence identity to SEQ ID NO: 14.In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 13, and a light chain comprising or consisting of an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 14. In some embodiments, the heavy and light chains are V as described herein. H CDR and V L In some embodiments, the V H The CDRs comprise the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In some embodiments, the V L The CDRs comprise or consist of the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiments, the heavy and light chains each can comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. In some embodiments, the heavy and light chains each can comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions in addition to the CDRs.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof is fully humanized. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. Methods for producing fully human antibodies are known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887 and 4,716,111, and WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741). Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see WO 98 / 24893, WO 92 / 01047, WO 96 / 34096, and WO 96 / 33735, and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598). Fully human antibodies recognizing a defined epitope can also be generated using a technique called "guided selection" (see Jespers et al., Biotechnology, 1988, 12, 899-903).

[0065] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody, which has binding specificities for at least two different antigens (i.e., one of its binding specificities is for BAI1 and the other binding specificity is for any other antigen, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a protein derived from a virus, an envelope protein encoded by a virus, a protein derived from a bacterium, or a bacterial surface protein, etc.).

[0066] In some embodiments, the antibody or antigen-binding fragment thereof is a derivatized antibody. For example, the derivatized antibody can be an antibody modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or the like. Any of numerous chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative can include one or more unnatural amino acids, such as using the technology of Ambrx (see Wolfson, Chem. Biol., 2006, 13, 1011-1012).

[0067] In some embodiments, the antibody or antigen-binding fragment thereof is derivatized through glycosylation. A suitable biantennary complex can be composed of a core structure having two N-acetylglucosamine (GlcNAc) residues, three mannose residues, and two GlcNAc residues linked β-1,2 to α-6 mannose and α-3 mannose to form two antennae. One or more fucose (Fuc) residues, galactose (Gal) residues, high-mannose glycan Man-5 or Man-9 residues, bisecting GlcNAc residues, and sialic acid (including N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA)) residues can be attached to the core. N-linked glycoforms may include G0 (a protein with a biantennary glycosylated core), G0F (fucosylated G0), G0F GlcNAc, G1 (a protein with a glycosylated core with one galactose residue), G1F (fucosylated G1), G2 (a protein with a glycosylated core with two galactose residues), and / or G2F (fucosylated G2). In some embodiments, the anti-BAI1 antibody has a G0F glycan.

[0068] The antibodies or antigen-binding fragments thereof of the present invention can be prepared by recombinantly expressing immunoglobulin light and heavy chain genes in host cells. To recombinantly express an antibody, one or more recombinant expression vectors containing DNA fragments encoding the antibody immunoglobulin light and heavy chains are transfected into host cells, allowing the light and heavy chains to be expressed in the host cells and, optionally, secreted into the medium in which the host cells are cultured, from which the antibody can be recovered. Standard recombinant DNA techniques can be used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells (see, for example, *Molecular Cloning: A Laboratory Manual, Second Edition, Sambrook, Fritsch, and Maniatis (eds.), Cold Spring Harbor, NY, 1989; *Current Protocols in Molecular Biology*, Ausubel et al., eds., Greene Publishing Associates, 1989; and U.S. Patent No. 4,816,397).

[0069] In some embodiments, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof described herein are generated by first obtaining DNA fragments encoding the light and heavy chain variable regions. These DNA fragments can be obtained, for example, by using the polymerase chain reaction (PCR) to amplify and modify germline DNA or cDNA encoding the light and heavy chain variable sequences. Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see the "VBASE" human germline sequence database; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., J. Mol. Biol., 1992, 22T, 116-198; and Cox et al., Eur. J. Immunol., 1994, 24, 827-836). A DNA fragment encoding the heavy or light chain variable region can be synthesized and used as a template for mutagenesis to generate variants as described herein using conventional mutagenesis techniques, or the DNA fragment encoding the variant can be synthesized directly.

[0070] The present disclosure provides a V HTCTGATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTAGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCCAATGCCCAGGGGTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 2, DNA) or UCUGAUGUGCA A nucleic acid molecule is provided that comprises or consists of the nucleotide sequence GCUUCAGGAGUCGGGACCUGGCCUGGUGAAACCUUCUCAGUCUCUGUCCCUCACCUGCACUGUCACUGGCUACUCAAUCACCAGUGAUUAUGCCUGGAACUGGAUCCGGCAGUUUCCAGGAAACAAACUGGAGUGGAUGGGCUACAUAAGCUACAGUGGUAGCACUAGCUACAACCCAUCUCUCAAAAGUCGAAUCUCUAUCACUCGAGACACAUCCAAGAACCAGUUCUUCCUGCAGUUGAAUUCUGUGACUACUGAGGACACAGCCACAUAUUACUGUGCCAAUGCCCAGGGGUAUGCUAUGGACUACUGGGGUCAAGGAACCUCAGUCACCGUCUCCUCA (SEQ ID NO: 3, RNA).

[0071] The present disclosure provides a V LGACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 5, DNA) or GACAUUGUGAU Also provided is a nucleic acid molecule comprising or consisting of the nucleotide sequence GACUCAGUCUCCAGCCACCCUGUCUGUGACUCCAGGAGAUAGAGUCUCUCUUUCCUGCAGGGCCAGCCAGAGUAUUAGCGACUACUUACACUGGUAUCAACAAAAAUCACAUGAGUCUCCAAGGCUUCUCAUCAAAUAUGCUUCCCAAUCCAUCUCUGGGAUCCCCUCCAGGUUCAGUGGCAGUGGAUCAGGGUCAGAUUUCACUCUCAGUAUCAACAGUGUGGAACCUGAAGAUGUUGGAGUGUAUUACUGUCAAAAUGGUCACAGCUUUCCAUUCACGUUCGGCUCGGGGACAAAGUUGGAAAUAAAA (SEQ ID NO: 6, RNA).

[0072] These DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. These manipulations involve linking the V to another DNA fragment encoding another protein (such as an antibody constant region or a flexible linker). H or V LThe phrase "operably linked," as used in this context, is intended to mean that the two DNA fragments are joined together so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0073] That V H The isolated DNA molecule encoding the V region is linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3 and optionally CH4). H A full-length heavy chain gene can be obtained by operably linking a DNA encoding the heavy chain constant region to a DNA fragment encoding the heavy chain constant region. The sequences of human heavy chain constant region genes are known in the art (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD. In some embodiments, the heavy chain constant region is the constant region of IgG1 or IgG4. For a Fab fragment heavy chain gene, the V constant region is operably linked to a separate DNA molecule encoding only the heavy chain CH1 constant region. H DNA encoding the gene can be operably linked.

[0074] V L The isolated DNA encoding the light chain constant region C L Another DNA molecule that encodes that V LA full-length light chain gene (and a Fab light chain gene) can be generated by operably linking a DNA encoding the V light chain constant region to another fragment encoding a flexible linker, e.g., a fragment encoding the amino acid sequence (Gly4Ser)3. The sequences of human light chain constant region genes are known in the art (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition (USDapartment of Health and Human Services, NIH Publication No. 91-3242)), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region. In some embodiments, the light chain constant region is a kappa constant region. To generate an scFv gene, the V light chain constant region is linked to another fragment encoding a flexible linker, e.g., a fragment encoding the amino acid sequence (Gly4Ser)3. H and V L DNA fragments encoding V are operably linked to form L Area and V H The V domains are expressed as a continuous single-chain protein connected by a flexible linker. H Sequence and V L The sequence is rendered expressible (see Bird et al., Science, 1988, 242, 423-426; Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883; and McCafferty et al., Nature, 1990, 348, 552-554).

[0075] To express the antibodies or antigen-binding fragments thereof described herein, DNA molecules encoding partial or full-length light and heavy chains (obtained as described herein) can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the phrase "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0076] The antibody genes can be inserted into an expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Prior to insertion of the light or heavy chain sequences, the expression vector can already contain antibody constant region sequences. For example, V H Sequence and V L One approach to converting the sequences into full-length antibody genes is to insert them into an expression vector that already encodes the heavy and light chain constant regions, and then H The segment is located in the C H operably linked to the segment(s), L The segment is located in the C LThe antibody chain segment is operably linked to the antibody segment. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (such as a signal peptide from a non-immunoglobulin protein).

[0077] In addition to the antibody chain genes, the recombinant expression vector can carry regulatory sequences that control the expression of the antibody chain genes in host cells. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be apparent to those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, and the like. Suitable regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), promoters and / or enhancers derived from simian virus 40 (SV40) (e.g., the SV40 promoter / SV40 enhancer), promoters and / or enhancers derived from adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyoma. Viral regulatory elements and sequences thereof are known in the art (see, eg, US Pat. Nos. 5,168,062, 4,510,245, and 4,968,615).

[0078] The recombinant expression vectors of the present disclosure may also have additional sequences, such as sequences that regulate replication of the vector in host cells (such as origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to drugs such as G418, puromycin, blasticidin, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (DHFR - The host cells may also contain a gene encoding the heavy and light chains (for use with methotrexate selection / amplification) and a neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to include a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0079] The antibodies or antigen-binding fragments thereof of the present invention can be expressed in either prokaryotic or eukaryotic host cells. In some embodiments, expression of the antibodies or antigen-binding fragments thereof can be carried out in eukaryotic cells, such as mammalian host cells, to ensure secretion of correctly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing the recombinant antibodies or antigen-binding fragments thereof of the present disclosure include Chinese hamster ovary (CHO) cells (including DHFR CHO cells (see Urlaub, Proc. Natl. Acad. Sci. USA, 1980, 77, 4216-4220) used with a DHFR selectable marker (see Kaufman, Mol. Biol., 1982, 159, 601-621)), NS0 myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to express the antibody in the host cell or secrete the antibody into the culture medium in which the host cell is grown. The antibody or antigen-binding fragment thereof can be recovered from the culture medium using standard protein purification methods. The host cell can also be used to produce portions of an intact antibody (such as Fab fragments or scFv molecules). It is understood that variations on the above procedures are within the scope of the present disclosure. For example, it may be desirable to transfect the host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody or antigen-binding fragment thereof described herein.

[0080] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to BAI1. Molecules expressed from such truncated DNA molecules are also included in the antibodies or antigen-binding fragments thereof described herein.

[0081] Additionally, bifunctional antibodies or antigen-binding fragments thereof, in which one heavy and light chain is an antibody of the present disclosure and the other heavy and light chain is specific for an antigen other than BAI1, can be generated by crosslinking an antibody of the present disclosure to a second antibody by standard chemical crosslinking methods. Bifunctional antibodies can also be generated by expressing nucleic acids engineered to encode the bifunctional antibody.

[0082] For recombinant expression of the antibodies or antigen-binding fragments thereof described herein, host cells can be co-transfected with two expression vectors of the present disclosure: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. Typically, the two vectors each contain a separate selectable marker. Alternatively, a single vector can be used that encodes both heavy and light chain polypeptides.

[0083] Once a nucleic acid encoding one or more portions of the antibody or antigen-binding fragment thereof having the desired CDR sequences has been generated, further modifications can be introduced into the coding sequence to generate, for example, nucleic acids encoding antibodies with different CDR sequences, antibodies with reduced affinity for Fc receptors, or antibodies of a different subclass.

[0084] The antibodies or antigen-binding fragments thereof described herein can also be produced by chemical synthesis (such as the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Variant antibodies or antigen-binding fragments thereof can also be produced using cell-free platforms (see Chu et al., Biochemia, 2001, 2).

[0085] Once the antibodies or antigen-binding fragments thereof described herein are produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (ion exchange chromatography, affinity chromatography, particularly affinity chromatography for Protein A, Protein G, or Protein L selection, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Additionally, the antibodies or antigen-binding fragments thereof described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0086] Once isolated, the antibody or antigen-binding fragment thereof can be further purified, if desired, such as by high performance liquid chromatography (see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology (Work and Burdon, eds., Elsevier, 1980)) or gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0087] The present disclosure provides a vector comprising any of the nucleic acid molecules described herein. The present disclosure also provides a prokaryotic host cell transformed with the vector. The present disclosure also provides a eukaryotic host cell transformed with the vector. In some embodiments, the eukaryotic host cell is a mammalian host cell.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an effector moiety. In some embodiments, the antibody or antigen-binding fragment thereof is modified by covalently attaching any type of molecule to the antibody or antigen-binding fragment thereof such that the covalent attachment does not interfere with binding to BAI1. In some embodiments, the effector moiety is a detectable label, a cytotoxic agent, a chemotherapeutic agent, or a nucleic acid molecule. The effector moiety can also be an anti-tumor agent, a drug, a toxin, a biologically active protein (such as an enzyme), another antibody or antibody fragment, a synthetic polymer, a natural polymer, a nucleic acid molecule, a radionuclide (such as radioactive iodine), a radioisotope, a chelated metal, a nanoparticle, or a reporter group (such as a fluorescent compound or a compound detectable by NMR spectroscopy or ESR spectroscopy).

[0089] In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to a cytotoxic agent, radionuclide, or drug moiety to modify a particular biological response. The effector moiety can be, for example, a toxin (such as abrin, ricin A, saporin, Pseudomonas exotoxin, diphtheria toxin, ethidium bromide, PE40, PE38, gelonin, RNAse, peptide nucleic acid (PNA), ribosome-inactivating protein type 1 or type 2 (RIP), pokeweed antiviral protein (PAP), bryodin, momordin, chemotherapeutic agents, and bouganin), a signaling molecule (such as alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, or can be a protein or polypeptide such as a biological response modifier, such as a thrombotic agent or angiogenesis inhibitor (such as angiostatin or endostatin), or a cytokine or growth factor (such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), or nerve growth factor (NGF)).

[0090] In some embodiments, the cytotoxic agent is a small molecule, prodrug, maytansinoid, or toxin. In some embodiments, the antibody or antigen-binding fragment thereof comprises 3 to 5 maytansinoid molecules per antibody or antigen-binding fragment thereof. In some embodiments, the maytansinoid is conjugated to the antibody or antigen-binding fragment thereof by a chemical linker selected from N-succinimidyl-3-(2-pyridyldithio)propionate, N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. In some embodiments, the cytotoxic agent is taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, or puromycin.

[0091] In some embodiments, the detectable label is a radioactive compound, a fluorescent compound, a chromophore, an enzyme, an imaging agent, a metal ion, or a substrate. In some embodiments, fluorescent moieties include, but are not limited to, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Useful enzyme labels include, but are not limited to, alkaline phosphatase, horseradish peroxidase, glucose oxidase, and the like.

[0092] In some embodiments, the effector moiety is an antimetabolite (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine and 5-fluorouracil, dacarbazine), an alkylating agent (such as mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C5, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), an anthracycline (such as daunorubicin (formerly daunomycin) and doxorubicin), an antibiotic (such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin or duocarmycin), or an antimitotic agent (such as vincristine and vinblastine).

[0093] In some embodiments, the radionuclide is 13 N, 18 F, 32 P, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 67 Cu, 77 Br, 80m Br, 82 Rb, 86 Y, 90 Y, 95 Ru, 97 Ru, 99m Tc, 103 Ru, 105 Ru, 111 In, 113m In, 113 Sn, 121m Te, 122m Te, 125m Te, 123 I, 124 I, 125 I, 126 I, 131 I, 133 I, 165 Tm, 167 Tm, 168 Tm, 177 Lu, 186 Re,188 Re, 195m Hg, 211 At, 212 Bi, 213 Bi and 225 Ac, but is not limited to these.

[0094] In some embodiments, the chemotherapeutic agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, methotrexate, vincristine, doxorubicin, tunicamycin, oligomycin, bortezomib, MG132, 5-fluorouracil, sorafenib, flavopiridol, gemcitabine, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, mitomycin, cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, procarbazine, or riboflavin. The drugs are benzodiazepine, etoposide, campatecin, bleomycin, idarubicin, daunorubicin, dactinomycin, distamycin A, ethidium, netropsin, auristatin, amsacrine, prodigiosin, bortezomib, pipendimol, tomaymycin, duocarmycin SA, plicamycin, mitoxantrone, asparaginase, vinblastine, vinorelbine, paclitaxel, docetaxel, CPT-11, Gleevec, erlotinib, gefitinib, ibrutinib, crizotinib, ceritinib, lapatinib, navitoclax, or regorafenib.

[0095] In some embodiments, the nucleic acid molecule is a 1-layer nucleic acid carrier, a 1.5-layer nucleic acid carrier, a 2-layer nucleic acid carrier, a 2.5-layer nucleic acid carrier, or a 3-layer nucleic acid carrier (e.g., those disclosed in WO 17 / 143156 and WO 17 / 143171).

[0096] Techniques for conjugating such effector moieties to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., pp. 623-53 (Robinson et al., eds., 1987)); Thorpe et al., Immunol. Rev., 1982, 62, 119-58; and Dubowchik et al., Pharmacology and Therapeutics, 1999, 83, 67-123).

[0097] In some embodiments, an antibody or antigen-binding fragment thereof of the invention can be fused by a covalent bond (peptide bond) to the amino acid sequence of another protein (or a portion thereof, e.g., at least 10, 20, or 50 amino acids of that portion of the protein) at the N-terminus, C-terminus, or internally of the antibody. The antibody or antigen-binding fragment thereof can be linked to the other protein at the N-terminus of the antibody's constant domain. Such fusions can be achieved using recombinant DNA techniques, for example, as described in WO 86 / 01533 and EP 0 392 745. In some embodiments, the effector moiety can increase in vivo half-life and / or enhance delivery of the antibody across epithelial barriers to the immune system. Examples of suitable effector moieties of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds (such as those described in WO 2005 / 117984).

[0098] In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to a small molecule toxin. In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to a dolastatin or a dolastatin peptide analog or derivative, such as an auristatin (see U.S. Pat. Nos. 5,635,483 and 5,780,588). The dolastatin or auristatin drug moiety can be attached to the antibody through its N-terminus, C-terminus, or internally (see WO 02 / 088172). Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, as disclosed in U.S. Pat. No. 7,498,298 (which discloses linkers and methods for preparing linker-conjugated monomethylvaline compounds, such as MMAE and MMAF).

[0099] Antibodies or antigen-binding fragments thereof can also be conjugated to liposomes for targeted delivery (see Park et al., Adv. Pharmacol., 1997, 40, 399-435 and Marty et al., Methods Molec. Med., 2004, 109, 389-401).

[0100] In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to a poly(ethylene glycol) (PEG) moiety. In some embodiments, the antibody or antigen-binding fragment thereof and PEG moiety can be attached through any available amino acid side chain or terminal amino acid functional group located on the antibody or antigen-binding fragment thereof, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids can be naturally present on the antibody or antigen-binding fragment thereof or can be engineered into fragments using recombinant DNA methods (see U.S. Pat. No. 5,219,996). Multiple sites can be used to attach two or more PEG moieties. A PEG moiety can be covalently attached through the thiol group of at least one cysteine ​​residue located on the antibody or antigen-binding fragment thereof. When a thiol group is used as the attachment site, an appropriately activated effector moiety can be used, such as a thiol-selective derivative, such as maleimide and cysteine ​​derivatives.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof can comprise a modified Fab' fragment that is PEGylated. The PEG moiety can be attached to a cysteine ​​in the hinge region. In some embodiments, the PEG-modified Fab' fragment has a maleimide group covalently attached to one thiol group in the modified hinge region. A lysine residue can be covalently attached to the maleimide group, and each of the amine groups on the lysine residue can be attached to a methoxypoly(ethylene glycol) polymer having a molecular weight of approximately 20,000 Da. Thus, the total molecular weight of PEG attached to the Fab' fragment can be approximately 40,000 Da.

[0102] In some embodiments, the antigen-binding fragment can be a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv fragment, an scFv-Fc fragment, a diabody fragment, or a minibody fragment. In some embodiments, the antigen-binding fragment can be a Fab fragment. In some embodiments, the antigen-binding fragment can be an F(ab')2 fragment. In some embodiments, the antigen-binding fragment can be an Fv fragment. In some embodiments, the antigen-binding fragment can be an scFv fragment. In some embodiments, the antigen-binding fragment can be an scFv-Fc fragment. In some embodiments, the antigen-binding fragment can be a diabody fragment. In some embodiments, the antigen-binding fragment can be a minibody fragment.

[0103] The present disclosure also provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a tonicity agent, a surfactant, a preservative, and / or a buffer system having a pH of about 4.0 to about 8.0. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, such as a combination therapeutic agent described herein. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition.

[0104] In some embodiments, the pharmaceutical compositions can be provided in unit dose form, containing a predetermined amount of an antibody or antigen-binding fragment thereof described herein per dose. Pharmaceutically acceptable carriers used in the pharmaceutical compositions can take a wide variety of forms, depending on the condition to be treated or the route of administration.

[0105] Pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof described herein can be prepared for storage as a lyophilized formulation or aqueous solution by mixing the antibody or antigen-binding fragment thereof of the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") typically used in the art, such as, for example, buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives.

[0106] Buffering agents help maintain pH in a range that approximates physiological conditions. Buffering agents can be present in concentrations ranging from about 2 mM to about 50 mM. Buffering agents suitable for use in the pharmaceutical compositions described herein include both organic and inorganic acids and their salts, such as citrate buffers (monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinic acid buffers (succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartaric acid buffers (tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumaric acid buffers (fumaric acid-monosodium fumarate mixtures, etc.), and the like. Examples of suitable buffers include: fumaric acid-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, gluconic acid buffers (gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures, etc.), oxalic acid buffers (oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactic acid buffers (lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can be used.

[0107] Preservatives can be added to the pharmaceutical compositions to reduce microbial growth and can be added in amounts ranging from about 0.2% to about 1% (w / v). Preservatives suitable for use with the pharmaceutical compositions described herein include, but are not limited to, phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (such as chloride, bromide, and iodide), hexamethonium chloride, alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0108] Tonicity agents (sometimes known as "stabilizers") can be added to ensure isotonicity of the liquid composition and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients whose function can range from bulking agents to additives that help solubilize the therapeutic agent or prevent denaturation or adhesion to the container wall. Typical stabilizers include polyhydric sugar alcohols (as listed above), amino acids (arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (including cyclitols such as inositol, lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, etc.), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (urea, Stabilizers can be selected from the group consisting of glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate, low molecular weight polypeptides (such as peptides of 10 residues or less), proteins (such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), monosaccharides (such as xylose, mannose, fructose, and glucose), disaccharides (such as lactose, maltose, and sucrose), trisaccharides (such as raffinose), and polysaccharides (such as dextran). The stabilizer can be present in an amount ranging from about 0.1 to about 10,000 parts by weight per part by weight of active protein.

[0109] Non-ionic surfactants or detergents (also known as "wetting agents") can also be added to pharmaceutical compositions to help solubilize the antibodies or antigen-binding fragments thereof of the present invention, protect the antibodies or antigen-binding fragments thereof described herein from agitation-induced aggregation, and allow the formulation to be exposed to shear stress without denaturing the protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), poloxamers (e.g., 184, 188), pluronic polyols, polyoxyethylene sorbitan monoethers (e.g., TWEEN™-20, TWEEN™-80). The non-ionic surfactant can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, e.g., about 0.07 mg / mL to about 0.2 mg / mL.

[0110] Additional excipients such as bulking agents (such as starch), chelating agents (such as EDTA), antioxidants (such as ascorbic acid, methionine, and vitamin E), and cosolvents may also be added to the pharmaceutical composition.

[0111] The present disclosure also provides pharmaceutical kits comprising the antibodies or antigen-binding fragments thereof of the invention, including antibody conjugates described herein. The pharmaceutical kits can be packages containing the antibodies or antigen-binding fragments thereof described herein (such as in either lyophilized form or in aqueous solution), one or more of the combination therapies described below, devices for administering the antibodies or antigen-binding fragments thereof (such as pens, needles, and / or syringes), and, if the antibodies are in lyophilized form, pharmaceutical-grade water or buffer for resuspending the antibodies or antigen-binding fragments thereof.

[0112] In some embodiments, each unit dose of the antibody or antigen-binding fragment thereof is packaged separately, and the kit can include one or more unit doses (e.g., two unit doses, three unit doses, four unit doses, five unit doses, eight unit doses, ten or more unit doses, etc.), in some embodiments, each of the one or more unit doses is contained in a syringe or pen.

[0113] Also included herein are diagnostic kits containing the antibodies or antigen-binding fragments thereof (including antibody conjugates) described herein. The diagnostic kit can be a package containing the antibodies or antigen-binding fragments thereof described herein (e.g., in either lyophilized form or aqueous solution) and one or more reagents useful for performing a diagnostic assay. If the antibody or antigen-binding fragment thereof is labeled with an enzyme, the kit can include substrates and cofactors required by the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (e.g., block buffer or lysis buffer), and the like can be included. In some embodiments, the antibodies or antigen-binding fragments thereof included in the diagnostic kit can be immobilized on a solid surface, or the kit includes a solid surface (e.g., a slide or plate) on which the antibodies or antigen-binding fragments thereof can be immobilized. The relative amounts of the various reagents can be varied to provide concentrations in the reagent solution that substantially optimize the sensitivity of the assay. In some embodiments, the antibodies or antigen-binding fragments thereof and one or more reagents can be supplied (individually or combined) as dry powders, typically lyophilized powders, with excipients that, when dissolved, provide a reagent solution of the appropriate concentration.

[0114] The present disclosure also provides a method for detecting a BAI1-expressing cell, the method comprising contacting the cell with an antibody or antigen-binding fragment thereof described herein and detecting the antibody or antigen-binding fragment thereof. In some embodiments, the cell is present in a biological sample obtained from a human, and the cell is contacted with the antibody or antigen-binding fragment thereof in vitro. In some embodiments, the cell is present in a human, and the cell is contacted with the antibody or antigen-binding fragment thereof in vivo.

[0115] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have high binding affinity to BAI1. In some embodiments, the antibodies or antigen-binding fragments thereof have high binding affinity to BAI1. on value or k A value), dissociation rate constant (k off value or k D value), affinity constant (K A value), dissociation constant (K D value) and / or IC 50 The value is a specific value.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof has a nucleotide sequence of at least about 10 10 M -1 , at least about 4 × 10 11 M -1 , at least about 10 11 M -1 , at least about 4 × 10 12 M -1 , at least about 10 12 M -1 , at least about 4 × 10 13 M -1 , at least about 10 13 M -1 , at least about 4 × 10 14 M -1 , at least about 10 14 M -1 , at least about 4 × 10 15 M -1 Or at least about 10 15 M -1 K A (k on / k off ), or any range (approximately 4 x 10 11 M -1 ~Approx. 4×10 13 M -1 Or about 4 x 10 12 M -1 ~Approx. 4×10 15 M -1 etc.) A and binds to BAI1.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof is -10 M or less, approximately 4 x 10 -11 M or less, about 10 -11 M or less, approximately 4 x 10 -12 M or less, about 10 -12 M or less, approximately 4 x 10 13 M or less, about 10 -13 M or less, approximately 4 x 10 14 M or less, about 10 -14 M or less, approximately 4 x 10 -15 M or less or about 10 -15 K below M D (k off / k on ), or any range (approximately 4 x 10 -11 M ~ approx. 4×10 -13 M or approximately 4 x 10 -12 M ~ approx. 4×10 -15 K in M ​​etc. D and binds to BAI1.

[0118] In some embodiments, the K D (k off / k on ) values ​​are determined by assays well known in the art, such as ELISA, isothermal titration calorimetry (ITC), fluorescence polarization assay or any other biosensor (such as BIAcore).

[0119] In some embodiments, the antibody or antigen-binding fragment thereof binds to BAI1 and has a concentration of less than about 0.02 nM, less than about 0.01 nM, less than about 0.005 nM, less than about 0.002 nM, less than about 0.001 nM, less than about 5 x 10 -4 Less than nM, approximately 2 × 10 -4 Less than nM, approximately 1 × 10 -4 Less than nM, approximately 5 × 10 -5 Less than nM, approximately 2 × 10 -5 Less than nM, approximately 1 × 10 -4 Less than nM, approximately 5 × 10 -6 Less than nM, approximately 2 × 10 -6 Less than nM, approximately 1 × 10 -6 Less than nM, approximately 5 × 10-7 Less than nM, approximately 2 × 10 -7 Less than nM or approximately 1 × 10 -7 IC below nM 50 or any range (approximately 0.02 nM to approximately 2 × 10) with any combination of the above values ​​as limit values. -5 nM or approximately 5 × 10 -5 nM ~ approx. 1×10 -7 IC of nM 50 It inhibits the binding of BAI1 to its ligand. 50 can be measured according to methods well known in the art, such as ELISA.

[0120] The antibodies or antigen-binding fragments thereof of the present invention, including antibodies modified, such as with biotinylation, horseradish peroxidase, or any other detectable moiety (including those described above), can be used for diagnostic purposes.

[0121] In some embodiments, the antibodies or antigen-binding fragments thereof can be used to purify or detect BAI1, including both in vitro and in vivo diagnostic methods. For example, the antibodies or antigen-binding fragments thereof can be used in immunoassays to qualitatively and quantitatively measure levels of BAI1 in biological samples or to identify the location, amount, behavior, and / or similar of BAI1 in animals. For example, measuring BAI1 levels using the antibodies or antigen-binding fragments thereof described herein can be used to, for example, 1) diagnose or determine an increased risk of developing cancer in a patient, 2) determine the patient's prognosis, including tumor stage and grade (particularly whether the cancer is metastatic or likely to be metastatic) and / or its potential susceptibility to BAI1 therapy, 3) determine the origin of the tumor, and / or 4) determine the effectiveness of a patient's treatment.

[0122] In some embodiments, the antibodies or antigen-binding fragments thereof can be used in conjunction with compound screening assays, e.g., to assess the effect of pharmaceutical agents on the expression and / or activity of the BAI1 gene product. Additionally, the antibodies or antigen-binding fragments thereof can be used in conjunction with gene therapy techniques, e.g., to assess the success of transfection of normal and / or engineered BAI1 expression.

[0123] The present disclosure also provides a method for diagnosing a neurological disease, comprising detecting the amount or activity of BAI1 expressed in nervous tissue or any tissue associated with a target organ other than the CNS, such as the lung, liver, kidney, spleen, etc. The diagnostic method can employ an antibody or antigen-binding fragment thereof conjugated to a diagnostic agent. The antibody or antigen-binding fragment thereof can be used diagnostically, for example, to detect BAI1 expression in specific cells, tissues, or serum, or to monitor the development or progression of an immune response as part of a clinical trial procedure, for example, to determine the effectiveness of a particular therapeutic regimen. Detection can be facilitated by binding the antibody or antigen-binding fragment thereof to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials (such as fluorescein and rhodamine and their derivatives), luminescent materials, bioluminescent materials, optical agents (such as porphyrin, anthraquinone, anthrapyrazole, perylenequinone, xanthene, cyanine, acridine, phenoxazine, and phenothiazine derivatives), radioactive materials, positron-emitting metals using various positron emission tomography techniques, non-radioactive paramagnetic metal ions (such as Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III), and V(IV)). The detectable substance can be bound or conjugated to the antibody or antigen-binding fragment thereof directly or indirectly through an intermediate (e.g., a linker known in the art) using techniques known in the art. Examples of enzyme labels include luciferases (firefly luciferase and bacterial luciferase).No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, monosaccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol, and examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131 I, 111 In or 99 Examples include, but are not limited to, Tc.

[0124] The present disclosure also provides a method for detecting BAI1 expression on cells, comprising contacting a biological sample from a patient with one or more of the antibodies or antigen-binding fragments thereof described herein (optionally conjugated to a detectable moiety) and detecting whether the sample is positive for BAI1 expression or whether expression is altered (decreased or increased) in the sample compared to a control sample. The biological sample may include biopsies of various tissues, including skin, muscle, breast, prostate, cervix, ovary, brain, testis, and lung. Examples of cells in the biological sample include tumor cells, skin cells, muscle cells, blood cells, ovarian cells, brain cells, prostate cells, breast cells, testicular cells, cervical cells, and lung cells. The biological sample may also be a biological fluid.

[0125] The presence of BAI1-expressing cells in a biological sample indicates the presence of cancer and may indicate metastasis, particularly when present in amounts greater than those in normal, healthy subjects. Loss of BAI1-expressing cells over time in a patient, particularly one undergoing treatment, indicates remission (i.e., successful treatment), while a lack of change in BAI1-expressing cell levels in a patient undergoing treatment indicates resistance to therapy and suggests that a different treatment strategy may be employed. Similarly, an increase in BAI1-expressing cells over time in a patient may indicate recurrence. Additionally, the imaging techniques described herein may be used to monitor tumor size and determine the effectiveness of treatment. In some embodiments, other cancer diagnostic assays may be performed to confirm the results obtained by the methods described herein.

[0126] In some embodiments, a biological sample (such as a tumor sample) can be obtained from a subject to determine the presence of BAI1-expressing cells. The number of BAI1-expressing cells can be correlated with tumor grade. In some embodiments, the number of BAI1-expressing cells in the biological sample is compared to the number of BAI1-expressing cells in a corresponding biological sample obtained from a healthy individual to determine modulation of BAI1-expressing cells in the tumor. A subject containing a tumor can be treated with a pharmaceutical agent to modulate the activity of BAI1-expressing cells to normal, healthy levels.

[0127] Diseases that can be diagnosed using the methods of the present invention include, but are not limited to, neuronal cancers such as primary brain tumors, including glioma (glioblastoma), meningioma, schwannoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid, and sarcoma, and intracranial metastases from other tumor sources. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to diagnose glioblastoma multiforme (GBM).

[0128] The present disclosure also provides a method of treating a BAI1-expressing cancer, comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described herein. In some embodiments, the method involves administering to a human patient with a solid tumor an amount of an antibody or antigen-binding fragment thereof described herein to produce a therapeutic effect.

[0129] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be administered to a patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intraarterial, intramuscular, intraocular, topical, regional, intrathecal, intraventricular, intraspinal, and intracranial routes. The most appropriate administration route in any given case will depend on the particular antibody, the subject, the nature and severity of the disease, and the physical condition of the subject. In some embodiments, the antibodies or antigen-binding fragments thereof can be formulated as an aqueous solution. In some embodiments, the antibodies or antigen-binding fragments thereof are administered intravenously or intracranially.

[0130] The antibodies or antigen-binding fragments thereof described herein can be used to treat a variety of neoplasms that express BAI1. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat cancers that express BAI1, such as sarcomas with characteristics of skeletal muscle tumors and skin tumors. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat neural cancers that express BAI1, such as brain tumors or small brain lesions (e.g., micrometastases) in patients. In some embodiments, the BAI1-expressing cancer is a neural cancer. In some embodiments, the neural cancer is a primary brain tumor, glioblastoma, glioma, meningioma, schwannoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid tumor, sarcoma, or intracranial metastasis from other tumor sources. In some embodiments, the neural cancer is a glioblastoma. In some embodiments, the glioblastoma is glioblastoma multiforme (GBM).

[0131] In some embodiments, the BAI1-expressing brain tumor is a GBM tumor comprising GBM tumor-initiating cells. In some embodiments, treatment with an antibody or antigen-binding fragment thereof described herein inhibits the proliferation of GBM tumor-initiating cells. In some embodiments, the antibody or antigen-binding fragment thereof further inhibits the self-renewal of GBM tumor-initiating cells. Inhibition of cell proliferation and / or self-renewal may ameliorate signs or symptoms of the disease. For example, such therapy may improve survival (overall survival and / or progression-free survival) and / or result in an objective clinical response (partial or complete response). In some embodiments, the antibody or antigen-binding fragment thereof is internalized by GBM tumor cells, thereby improving the therapeutic efficacy of the antibody or antigen-binding fragment thereof in killing GBM tumor cells to which it binds. In some embodiments, the antibody or antigen-binding fragment thereof functions as an antagonist of BAI1 biological activity and, additionally, may be used as a method for inhibiting aberrant BAI1 activity.

[0132] In some embodiments, the antibodies or antigen-binding fragments thereof are useful for treating non-neural tumors that express BAI1, including cancers and benign tumors. Cancers likely to respond to treatment with the antibodies or antigen-binding fragments thereof described herein include cancers that overexpress BAI1. In some embodiments, cancers likely to respond to treatment with the antibodies or antigen-binding fragments thereof described herein include epithelial cell cancers. In some embodiments, cancers likely to respond to treatment with the antibodies or antigen-binding fragments thereof described herein include, but are not limited to, breast cancer, ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, ovarian cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, thymic cancer, head and neck cancer, and colorectal cancer. The cancer may be newly diagnosed or untreated, and may be recurrent, refractory, or relapsed refractory cancer, or a metastatic form of a solid tumor.

[0133] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in the treatment of hematological malignancies that express BAI1, including, but not limited to, myeloma (such as multiple myeloma), lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma), leukemia (such as chronic lymphocytic leukemia, acute myeloid leukemia, and acute lymphocytic leukemia), and myelodysplastic syndrome. In some embodiments, the method comprises administering an antibody or antigen-binding fragment thereof described herein to a human patient with a hematological malignancy to produce a therapeutic effect.

[0134] In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein is repeated after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration can be at the same dose or at a different dose. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. For example, according to a particular administration regimen, a patient can receive anti-BAI1 therapy for an extended period of time, such as 6 months or a year or more. The amount of an antibody or antigen-binding fragment thereof described herein administered to a patient is a therapeutically effective amount. As used herein, a "therapeutically effective" amount of an antibody or antigen-binding fragment thereof described herein can be administered as a single dose or over a treatment regimen (e.g., over 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, a year, or more). Exemplary treatment regimens are further described herein. Treating a disease includes treating a patient already diagnosed with any form of the disease at any clinical stage or clinical manifestation, delaying the onset, progression, progression or worsening of symptoms or signs of the disease, and / or preventing the disease and / or reducing the severity of the disease.

[0135] Treating cancer in patients with the antibodies or antigen-binding fragments thereof described herein may demonstrate any clinical benefit compared to no treatment (if appropriate) or known standard of care. Clinical benefit can be assessed by any method known to one of skill in the art. In some embodiments, clinical benefit is assessed based on the objective response rate (ORR) (as determined using RECIST version 1.1), duration of response (DOR), progression-free survival (PFS), and / or overall survival (OS). In some embodiments, therapeutic benefit is indicated by a complete response. In some embodiments, therapeutic benefit is indicated by a partial response. In some embodiments, therapeutic benefit is indicated by stable disease. In some embodiments, therapeutic benefit is indicated by an increase in overall survival. In some embodiments, therapeutic benefit may be considered an improvement in time to disease progression and / or an improvement in symptoms or quality of life. In some embodiments, therapeutic benefit may not result in an extended period of disease control, but rather may result in a significant reduction in symptom burden, resulting in an improved quality of life. As will be apparent to one of skill in the art, therapeutic effects may be observed using the antibodies or antigen-binding fragments thereof described herein alone (monotherapy) or as an adjunct to or in conjunction with other anti-cancer therapies and / or targeted or non-targeted anti-cancer agents.

[0136] In some embodiments, therapeutic efficacy can be assessed using standard clinical tests designed to measure response to new cancer therapies. The therapeutic efficacy of the antibodies or antigen-binding fragments thereof described herein can be assessed using one or a combination of the following tests: 1) Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1, 2) immune-related RECIST (irRECIST), 3) Eastern Cooperative Oncology Group (ECOG) Performance Status, 4) immune-related response criteria (irRC), 5) disease evaluable by assessment of tumor antigens, 6) validated patient-reported outcome measures, and / or 7) Kaplan-Meier estimators for overall survival and progression-free survival.

[0137] The present disclosure also provides a method of combination therapy comprising administering to a patient at least two agents, wherein a first agent is an antibody or antigen-binding fragment thereof described herein and a second agent is a combination therapeutic agent. The antibody or antigen-binding fragment thereof described herein and the combination therapeutic agent can be administered simultaneously, sequentially, or separately. The combination therapy method may enhance the effect of the additive.

[0138] In the methods of the present disclosure, the antibodies or antigen-binding fragments thereof and the combination therapeutic agents described herein can be administered simultaneously or sequentially, with overlapping time periods. The antibodies or antigen-binding fragments thereof and the combination therapeutic agents described herein are administered sequentially if they are administered to a patient on the same day, e.g., the same patient visit day. Sequential administration can be performed 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours apart. In contrast, the antibodies or antigen-binding fragments thereof and the combination therapeutic agents described herein can be administered separately if they are administered to a patient on different days, e.g., the antibodies or antigen-binding fragments thereof and the combination therapeutic agents can be administered 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month apart. In the methods of the present disclosure, administration of the antibodies or antigen-binding fragments thereof described herein can precede or follow administration of the combination therapeutic agents. In some embodiments, the antibodies or antigen-binding fragments thereof and the combination therapeutic agents described herein are administered in an overlapping time period, followed by alternating administration of the antibodies or antigen-binding fragments thereof and the combination therapeutic agents for a second period.

[0139] In some embodiments, the combination therapeutic agent is a chemotherapeutic agent, an angiogenesis inhibitor, an antirheumatic drug, an anti-inflammatory agent, a radiotherapeutic agent, an immunosuppressant, or a cytotoxic agent. The antibodies or antigen-binding fragments thereof described herein can be used in combination with conventional cancer therapies (e.g., surgery, radiation therapy, chemotherapy, or a combination thereof). In some embodiments, the method further comprises one or both of surgically removing tumor cells and / or administering radiation therapy. In some embodiments, other therapeutic agents useful in combination tumor therapy with the antibodies or antigen-binding fragments thereof described herein include antagonists (e.g., antibodies) of other factors involved in tumor growth (e.g., HER2, HER3, HER4, VEGF, or TNF-α). In some embodiments, administering one or more cytokines to the patient may also be beneficial for cancer treatment. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered in combination with a growth inhibitory agent.

[0140] For the treatment of cancer, it may be appropriate to use anti-inflammatory agents in combination with the antibodies or antigen-binding fragments thereof described herein, including, but not limited to, acetaminophen, diphenhydramine, meperidine, dexamethasone, pentasa, mesalazine, asacol, codeine phosphate, benorylate, fenbufen, naprosyn, diclofenac, etodolac, indomethacin, aspirin, and ibuprofen.

[0141] For the treatment of cancer, chemotherapeutic agents can be used in combination with the antibodies or antigen-binding fragments thereof described herein. Chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins (such as cytotoxins or cytotoxic agents), including any agent detrimental to cell viability, and pharmaceutical agents containing chemotherapeutic compounds, and liposomes or other vesicles. Examples of suitable chemotherapeutic agents include 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, anti-α5β1 integrin antibodies, alkylating agents, allopurinol sodium, altretamine, amifostine, anastrozole, anthramycin (AMC), antimitotic agents, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, live BCG (intravesical), betamethasone sodium phosphate and betamethasone acetate, and bicalutamide. , bleomycin sulfate, busulfan, calcium leucovorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), chlorambucil, cisplatin, cladribine, colchicine, conjugated estrogens, cyclophosphamide, cytarabine, cytochalasin B, cytoxan, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, daunorubicin citrate, denileukin diftitox, dexrazoxane, dibromomannitol, dihydroxyanthracene dione, docetaxel, dolasetron mesylate, doxorubicin, dronabinol, E.coli L-asparaginase, volociximab, emetine, epoetin-α, Erwinia L-asparaginase, esterified estrogens, estradiol, estramustine sodium phosphate, ethidium bromide, ethinyl estradiol, etidronate, etoposide citrovorum factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine, glucocorticoids, goserelin acetate, gramicidin D, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha-2b, irinotecan, letrozole, leucovorin calcium, leuprolide acetate, levamisole, lidocaine, lomustine, maytansinoids, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate Examples of chemotherapeutic agents include, but are not limited to, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondansetron, paclitaxel, pamidronate disodium, pentostatin, pilocarpine, plicamycin, poriflozan 20 carmustine implant, porfimer sodium, procaine, procarbazine, propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, taxol, teniposide, testolactone, tetracaine, thiotepa, chlorambucil, thioguanine, thiotepa, topotecan, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate, or any salt thereof. In some embodiments, the method comprises administering at least one chemotherapeutic agent to the patient.

[0142] Any angiogenesis inhibitor can be used in combination with the antibodies or antigen-binding fragments thereof described herein. In some embodiments, the angiogenesis inhibitor is a VEGF antagonist or another VEGF receptor antagonist (such as a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, a low molecular weight inhibitor of VEGFR tyrosine kinase, and combinations thereof). Alternatively or additionally, an anti-VEGF antibody can be co-administered to the patient.

[0143] The treatment regimen administered to a patient can vary depending on the patient's age, weight, and disease state. The treatment regimen can last from 2 weeks to indefinitely. In some embodiments, the treatment regimen lasts for about 2 weeks to about 6 months, about 3 months to about 5 years, about 6 months to about 1 year or about 2 years, about 8 months to about 18 months, etc. The treatment regimen can be a fixed dose regimen or a regimen with multiple dose changes.

[0144] The dosage of the antibodies or antigen-binding fragments thereof described herein can depend on a variety of factors, including, but not limited to, the particular type of solid tumor being treated, the stage of the solid tumor being treated, the method of administration, the frequency of administration, the desired therapeutic effect, and other parameters (such as the patient's age, weight, and other characteristics). Determination of an effective dosage to produce a therapeutic effect for a given method and frequency of administration is within the ability of one of ordinary skill in the art. An effective dosage to produce a therapeutic effect may be initially estimated from in vivo animal models or clinical trials. Suitable animal models for a wide range of diseases are known in the art. The antibodies or antigen-binding fragments thereof described herein may be administered by any route appropriate to the condition to be treated.

[0145] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are supplied as a lyophilized powder in a vial. The vial can contain about 100 mg, about 125 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, or about 400 mg of the antibody or antigen-binding fragment. Prior to administration, the lyophilized powder can be reconstituted with sterile water for injection (SWFI) or other suitable vehicle to provide a solution containing the antibodies or antigen-binding fragments described herein. In some embodiments, the resulting reconstituted solution is further diluted with saline or other suitable infusion vehicle and administered, for example, via IV infusion, twice every 7 days, once every 7 days, once every 14 days, once every 21 days, once every 28 days, once every 35 days, once every 42 days, once every 49 days, or once every 56 days. In some embodiments, during the first cycle, the infusion is administered over 90 minutes. In some embodiments, subsequent infusions are administered over 60 minutes.

[0146] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered as an IV infusion at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg once every 7 days. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered as an IV infusion at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg once every 14 days. In some embodiments, an antibody or antigen-binding fragment thereof described herein is administered as an IV infusion at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg once every 21 days. In some embodiments, an antibody or antigen-binding fragment thereof described herein is administered as an IV infusion at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg once every 28 days.

[0147] When administered as an adjunct to or in conjunction with other agents, such as other chemotherapeutic agents, the antibodies or antigen-binding fragments described herein can be administered on the same schedule as the other agent(s) or on a different schedule. When administered on the same schedule, the antibodies or antigen-binding fragments described herein can be administered before the other agent(s), after the other agent(s), or overlapping in time with the other agent(s). In some embodiments, when the antibodies or antigen-binding fragments described herein are administered as an adjunct to or in conjunction with standard of care, the antibodies or antigen-binding fragments can be initiated before the initiation of the standard of care therapy, for example, one day, several days, one week, several weeks, one month, or even several months before initiating the standard of care therapy. In some embodiments, when the antibodies or antigen-binding fragments described herein are administered as an adjunct to or in conjunction with standard of care, the antibodies or antigen-binding fragments described herein can be initiated after initiation of the standard of care therapy, for example, one day, several days, one week, several weeks, one month, or even several months after initiating the standard of care therapy.

[0148] The administration schedule for subsequent administrations can vary from once every six months to daily, depending on a number of clinical factors, including the type of disease, the severity of the disease, and the patient's sensitivity to the antibodies or antigen-binding fragments thereof described herein.

[0149] The present disclosure also provides a method of treating posterior capsule opacification (PCO), comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the eye.

[0150] The present disclosure also provides a method of treating fibrosis, comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof is administered to an organ. In some embodiments, the organ is a kidney or a lung.

[0151] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in methods to isolate and / or purify cells, such as myofibroblast precursors, by selecting cells that bind to the antibodies or antigen-binding fragments thereof. Such cells can rapidly migrate to wounds in the skin, lens, retina, and brain, thereby aiding in wound healing. Such cells have the potential to develop into contractile myofibroblasts. Human adipose tissue contains a large number of such cells (i.e., BAI1). + cells) (unpublished results). + The cells can be administered to humans or transplanted into slow-healing or non-healing wounds (such as diabetic ulcers, pressure ulcers and severe surgical excisions) to promote wound closure.

[0152] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in methods for isolating neuroprotective cells by selecting cells that bind to the antibodies or antigen-binding fragments thereof. Cells that express BAI1 increase in number in response to retinal and brain injury. Such cells can be administered to the retina and brain to reduce neuronal death. BAI1 + The cells and / or the molecule(s) they produce can be injected into the vitreous of the eye or into the vasculature of the brain after injury.

[0153] The following exemplary embodiments are provided:

[0154] Embodiment 1. An antibody or antigen-binding fragment thereof that binds to the human adhesion G protein-coupled receptor B1 (BAI1) protein, comprising the amino acid sequence set forth in SEQ ID NO: 7, and H The first complementarity-determining region (CDR) in the V H - CDR1) and the amino acid sequence set forth in SEQ ID NO: 8, H The second CDR in the V H -CDR2) and the amino acid sequence set forth in SEQ ID NO: 9, H The third CDR in the V H-CDR3) and a variable light (V L ) chain, L -CDR1) and the amino acid sequence set forth in SEQ ID NO: 11, L The second CDR in the V L -CDR2) and the amino acid sequence set forth in SEQ ID NO: 12, L The third CDR in the V L -CDR3) or an antigen-binding fragment thereof.

[0155] Embodiment 2. V comprising the amino acid sequence set forth in SEQ ID NO: 1 H 2. The antibody or antigen-binding fragment thereof of embodiment 1, comprising a chain.

[0156] Embodiment 3. V comprising the amino acid sequence set forth in SEQ ID NO:4 L 2. The antibody or antigen-binding fragment thereof of embodiment 1, comprising a chain.

[0157] Embodiment 4. V comprising the amino acid sequence set forth in SEQ ID NO: 1 H a V chain comprising the amino acid sequence set forth in SEQ ID NO:4; L 2. The antibody or antigen-binding fragment thereof of embodiment 1, comprising a chain.

[0158] Embodiment 5. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the antibody is an IgM antibody or an IgG antibody.

[0159] Embodiment 6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the antibody is an IgM antibody.

[0160] Embodiment 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the antibody is an IgG antibody.

[0161] Embodiment 8. The antibody or antigen-binding fragment thereof of embodiment 7, wherein the antibody is an IgG1 antibody.

[0162] Embodiment 9. The antibody or antigen-binding fragment thereof of embodiment 7, wherein the antibody is an IgG1 G1m17 allotype antibody.

[0163] Embodiment 10. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, which is humanized.

[0164] Embodiment 11. The humanized antibody or antigen-binding fragment thereof of embodiment 10, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0165] Embodiment 12. The humanized antibody or antigen-binding fragment thereof of embodiment 10, comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0166] Embodiment 13. The humanized antibody or antigen-binding fragment thereof of embodiment 10, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0167] Embodiment 14. The antibody or antigen-binding fragment thereof of any one of embodiments 1 to 13, which is conjugated to an effector moiety.

[0168] Embodiment 15. The antibody or antigen-binding fragment thereof of embodiment 14, wherein the effector moiety is a detectable label, a cytotoxic agent, a chemotherapeutic agent, or a nucleic acid molecule.

[0169] Embodiment 16. The antibody or antigen-binding fragment thereof of embodiment 15, wherein the detectable label is a radioactive compound, a fluorescent compound, a chromophore, an enzyme, an imaging agent, a metal ion, or a substrate.

[0170] Embodiment 17. The antibody or antigen-binding fragment thereof of embodiment 15, wherein the cytotoxic agent is a small molecule, a prodrug, a maytansinoid, or a toxin.

[0171] Embodiment 18. The antibody or antigen-binding fragment thereof of embodiment 15, comprising 3 to 5 maytansinoid molecules per antibody or antigen-binding fragment thereof.

[0172] Embodiment 19. The antibody or antigen-binding fragment thereof of embodiment 18, wherein the maytansinoid is conjugated to the antibody or antigen-binding fragment thereof by a chemical linker selected from N-succinimidyl-3-(2-pyridyldithio)propionate, N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

[0173] Embodiment 20. The antibody or antigen-binding fragment thereof of embodiment 15, wherein the cytotoxic agent is taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, or puromycin.

[0174] Embodiment 21. The chemotherapeutic agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, methotrexate, vincristine, doxorubicin, tunicamycin, oligomycin, bortezomib, MG132, 5-fluorouracil, sorafenib, flavopiridol, gemcitabine, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, mitomycin, cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, procarbazine, etoposide, campatecin. , bleomycin, idarubicin, daunorubicin, dactinomycin, distamycin A, ethidium, netropsin, auristatin, amsacrine, prodigiosin, bortezomib, pipendimol, tomaymycin, duocarmycin SA, plicamycin, mitoxantrone, asparaginase, vinblastine, vinorelbine, paclitaxel, docetaxel, CPT-11, Gleevec, erlotinib, gefitinib, ibrutinib, crizotinib, ceritinib, lapatinib, navitoclax or regorafenib.

[0175] Embodiment 22. The antibody or antigen-binding fragment thereof of embodiment 15, wherein the nucleic acid molecule is a 1-layer nucleic acid carrier, a 1.5-layer nucleic acid carrier, a 2-layer nucleic acid carrier, a 2.5-layer nucleic acid carrier, or a 3-layer nucleic acid carrier.

[0176] Embodiment 23. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 22, wherein the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv fragment, an scFv-Fc fragment, a diabody fragment, or a minibody fragment.

[0177] Embodiment 24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier.

[0178] Embodiment 25. The pharmaceutical composition of embodiment 24, which is a liquid pharmaceutical composition.

[0179] Embodiment 26. The pharmaceutical composition of embodiment 24 or embodiment 25, further comprising an isotonicity agent, a surfactant, a preservative, and / or a buffer system having a pH of about 4.0 to about 8.0.

[0180] Embodiment 27. V of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9 H An isolated nucleic acid molecule encoding a chain, the nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:2 or SEQ ID NO:3.

[0181] Embodiment 28. V of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9 L An isolated nucleic acid molecule encoding a chain, the nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0182] Embodiment 29. A vector comprising the nucleic acid molecule of embodiment 27 or embodiment 28.

[0183] Embodiment 30. A prokaryotic host cell transformed with the vector of embodiment 29.

[0184] Embodiment 31. A eukaryotic host cell transformed with the vector of embodiment 29.

[0185] Embodiment 32. The eukaryotic host cell of embodiment 31, which is a mammalian host cell.

[0186] Embodiment 33. A method for detecting BAI1-expressing cells, comprising contacting the cells with an antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 23, and detecting the antibody or antigen-binding fragment thereof.

[0187] Embodiment 34. The method of embodiment 33, wherein the cells are present in a biological sample obtained from a human, and the cells are contacted with the antibody or antigen-binding fragment thereof in vitro.

[0188] Embodiment 35. The method of embodiment 33, wherein the cell is present in a human and the cell is contacted with the antibody or antigen-binding fragment thereof in vivo.

[0189] Embodiment 36. A method for treating a BAI1-expressing cancer, comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 23.

[0190] Embodiment 37. The method of embodiment 36, wherein the BAI1-expressing cancer is a neural cancer.

[0191] Embodiment 38. The method of embodiment 37, wherein the neural cancer is a primary brain tumor, glioblastoma, glioma, meningioma, schwannoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid, sarcoma, or intracranial metastasis from other tumor sources.

[0192] Embodiment 39. The method of embodiment 38, wherein the neural cancer is glioblastoma.

[0193] Embodiment 40. The method of any one of embodiments 36 to 39, wherein the antibody or antigen-binding fragment thereof is administered intravenously or intracranially.

[0194] Embodiment 41 The method of any one of embodiments 36-40, further comprising administering to the patient at least one chemotherapeutic agent.

[0195] Embodiment 42. The method of any one of embodiments 36-41, further comprising one or both of surgically removing tumor cells and / or administering radiation therapy.

[0196] Embodiment 43. A method of treating posterior capsule opacification (PCO), comprising administering to a human patient in need thereof the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 23.

[0197] Embodiment 44 The method of embodiment 43, wherein the antibody or antigen-binding fragment thereof is administered to the eye.

[0198] Embodiment 45. A method of treating fibrosis, comprising administering to a human patient in need thereof the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 23.

[0199] Embodiment 46 The method of embodiment 45, wherein the antibody or antigen-binding fragment thereof is administered to an organ.

[0200] Embodiment 47. The method of embodiment 46, wherein the organ is a kidney or a lung.

[0201] Embodiment 48. A method of promoting wound healing, comprising administering to a human patient in need thereof the antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 23.

[0202] Embodiment 49. The method of embodiment 48, wherein the wound is in the skin, the lens of the eye, the retina, or the brain.

[0203] Embodiment 50. The method of embodiment 48, wherein the wound is a diabetic ulcer or a pressure ulcer.

[0204] Embodiment 51. A method of promoting wound healing, comprising administering to a human patient in need thereof BAI1-expressing cells.

[0205] In order to more efficiently understand the subject matter disclosed herein, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the claimed subject matter in any way. Except where otherwise indicated, throughout these examples, molecular cloning reactions and other standard recombinant DNA techniques were performed using commercially available reagents according to the methods described in Maniatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989). As used herein, immunoglobulin amino acid residue numbering is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. [Example]

[0206] Example 1: Identification of the G8 (BAI1) antigen The membrane proteome array (MPA) is a platform for profiling the specificity of antibodies and other ligands targeting human membrane proteins. MPA can be used to determine antibody-target specificity, deconvolute targets of orphan antibodies, and characterize the target profiles of biosimilar candidates. Flow cytometry is used to directly detect antibody binding to membrane proteins expressed in eukaryotic cells, such as human HEK-293 cells. Consequently, all MPA targets are designed to have native conformation and appropriate post-translational modifications. The workflow of this process is shown in Figure 1. The G8 antibody was tested for reactivity against an MPA library of over 4,500 human membrane proteins, including GPCRs, ion channels, and transporters. Identified targets were validated in a secondary screen to confirm reactivity.

[0207] To determine optimal antibody concentrations and minimize background reactions, various concentrations of G8 lot 3 / 21 / 13 and G8 lot PS30170160 were tested using HEK-293T (human) cells expressing either the Protein A and Protein G constructs or vector alone. These experiments were performed in a 384-well format using a single dilution of the secondary antibody (Figures 2A and 2B, Table 1). Data from the assay setup experiments were used to determine optimal screening conditions for high-throughput immunodetection (Table 2). Briefly, to optimize conditions for antibody detection, cells were transfected in a 384-well format with either the Protein A and Protein G expression constructs (positive control for MAb binding) or vector alone (negative control), and then expression in the cells was detected using a high-throughput immunofluorescence flow cytometry assay. Serial dilutions (starting at 4 μg / ml) of each test and control MAb were tested for immunoreactivity against cells expressing Protein A and Protein G or vector alone. IgM was found not to react strongly with the positive control construct. Consequently, these experiments were used to determine conditions with low background activity, which indicates that high concentrations can be detected without blocking potential hits. [Table 1] [Table 2]

[0208] Determining antibody-binding targets To identify antibody binding targets, 5,300 distinct membrane proteins were each expressed in HEK-293T cells in individual wells arrayed in a 384-well plate. The cells were then matrixed by pooling individual columns and rows from each 384-well plate. The resulting MPA matrix was probed with G8 lot 3 / 21 / 13 and G8 lot PS30170160 at a concentration of 30 μg / ml and then detected with fluorescently labeled secondary antibodies. Positive controls (constructs expressing known targets) and negative controls (empty vector) were used to validate the fluorescence readings obtained from each experimental plate. Each individual membrane protein target was assigned a value corresponding to the binding values ​​of its unique row and column pools. The resulting binding values ​​(including row and column components) were normalized and transformed to obtain a single numerical value for antibody binding to each target protein (normalized target binding value). Targets were subsequently identified by detecting antibody binding to overlapping column and row pools from the same plate, thereby enabling specific deconvolution (Figures 3A and 3B, Table 3). Antibody binding was detected by flow cytometry using fluorescent secondary antibodies. [Table 3]

[0209] The screening yielded binding targets that were confirmed in a second flow cytometry assay using serial dilutions of the antibodies (Figures 4A and 4B). Accordingly, HEK-293T cells were transfected with either the target-expressing plasmid construct or the vector alone. Serial dilutions of each MAb were tested for immunoreactivity against cells expressing the target protein or the vector alone. Finally, the identity of all targets was reconfirmed by sequencing.

[0210] Example 2: Identification of the heavy and light chains of G8 by RACE To identify the nucleic acid molecules encoding the heavy and light chains of the G8 antibody, rapid amplification of cDNA ends (RACE) reactions were performed. RACE PCR reaction samples were analyzed on an agarose gel to visualize the amplified DNA fragments. Qualified antibody variable region DNA fragments ranging in size from 500 to 700 base pairs were amplified (Figure 5).

[0211] PCR-amplified DNA fragments from 24 clones were recovered from agarose gels and sequenced. CDRs were identified using the VBASE2 tool.

[0212] Example 3: Construction, production and characterization of humanized anti-BAI1 IgG The expression constructs for G8 IgG were cloned into a high-expression mammalian vector. Each DNA construct for G8 IgG was scaled up for transfection and sequence verified. 0.03 liters of transient production was completed in HEK293 cells (Tuna293™ process). Conditioned medium from the transient production run was harvested and clarified by centrifugation and filtration. The supernatant was applied to a Protein A column pre-equilibrated with binding buffer. OD 280 The wash buffer was passed through the column until the OD of each fraction was measured (NanoDrop, Thermo Scientific) and reached 0. The target protein was eluted with a low pH buffer, fractions were collected, and the OD of each fraction was measured. 280 The OD values ​​were recorded. The fractions containing the target protein were pooled and filtered through a 0.2 μm membrane filter. The protein concentration was determined by OD 280 From the calculated values ​​and the calculated extinction coefficient, 2.92 mg of G8 IgG was obtained. The data are summarized in Table 4. Final protein yield, aliquots, and analysis. [Table 4] [Table 5]

[0213] CE-SDS analysis was then performed using a LabChip GXII (Perkin Elmer). Endotoxin determination was performed on purified product samples using a chromogenic Limulus amebocyte lysate method (Endosafe-MCS, Charles River). Samples were run in duplicate. The experiment confirmed that all samples met the requirement of less than 1 EU / mg (Table 4). SE-UPLC analysis was performed using an SEC standard (MEDNA, catalog no. Y3101) as a protein size reference. All proteins were observed to contain >99% monomer. Mass spectrometry intact mass QC was performed, and the observed molecular weights of the IgGs were within the expected range.

[0214] To measure the relative affinity of G8 IgM and anti-BAI1 IgG (also referred to herein as "G8 IgG"), ELISA assays were performed with both G8 IgG and G8 IgM. Briefly, plates were coated with 2 μg / ml human BAI1 overnight at 4°C and blocked with 1% BSA in PBS for 1 hour at room temperature. The first incubation was performed for 1 hour at room temperature with either a 1:2 serial dilution of G8 IgM (starting at 20 μg / ml) or a 1:2 serial dilution of G8 IgG (anti-BAI1 IgG) (starting at 100 μg / ml). The second incubation was performed for 1 hour at room temperature with either HRP-conjugated anti-mouse IgM Fc (G8 IgM) or HRP-conjugated anti-human Fc (G8 IgG). The assay was developed with 3,3',5,5'-tetramethylbenzidine (TMB) by incubation for 15 minutes and stopped with 1 M HCl. Each assay was performed in duplicate. The results are shown in Figure 6. Both the G8 IgG and G8 IgM control samples showed positive ELISA signals.

[0215] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the above description. Such modifications are intended to be included within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. patents, non-U.S. patents, patent application publications, international patent application publications, GenBank accession numbers, etc.) is hereby incorporated by reference in its entirety.

Claims

1. An antibody or antigen-binding fragment thereof that binds to human adhesion G protein-coupled receptor B1 (BAI1) protein, A variable-weight (V) antibody comprising the amino acid sequence set forth in SEQ ID NO:

7. H The first complementarity determining region (CDR) in the V H - CDR1), and V comprising the amino acid sequence set forth in SEQ ID NO: 8 H The second CDR in the V H - CDR2), and V comprising the amino acid sequence set forth in SEQ ID NO:9 H The third CDR in the V H - CDR3), and A variable light (V) comprising the amino acid sequence set forth in SEQ ID NO:

10. L The first CDR (V L - CDR1), and V comprising the amino acid sequence set forth in SEQ ID NO: 11 L The second CDR in the V L - CDR2), and V comprising the amino acid sequence set forth in SEQ ID NO: 12 L The third CDR in the V L - CDR3), and The antibody or antigen-binding fragment thereof comprising:

2. V comprising the amino acid sequence set forth in SEQ ID NO: 1 H The antibody or antigen-binding fragment thereof of claim 1, comprising a chain.

3. V comprising the amino acid sequence set forth in SEQ ID NO:4 L 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, comprising a chain.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is an IgM antibody or an IgG antibody. An antibody or antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is humanized.

6. The humanized antibody or antigen-binding fragment thereof according to claim 5, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:

13.

7. 7. The humanized antibody or antigen-binding fragment thereof according to claim 5 or claim 6, comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO:

14.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, conjugated to an effector moiety.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the effector moiety is a detectable label, a cytotoxic agent, or a chemotherapeutic agent.

10. The antibody or antigen-binding fragment thereof according to claim 9, comprising 3 to 5 maytansinoid molecules per antibody or antigen-binding fragment thereof.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, an Fv fragment, an scFv fragment, an scFv-Fc fragment, a diabody fragment, or a minibody fragment.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

14. the nucleic acid sequence encoding the VH chain of the antibody or antigen-binding fragment thereof comprises the nucleotide sequence set forth in SEQ ID NO:2 or SEQ ID NO:3, L 14. The isolated nucleic acid molecule of claim 13, wherein the nucleic acid sequence encoding the chain comprises the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:

6.

15. A vector comprising the nucleic acid molecule of claim 13 or claim 14.

16. A host cell transformed with the vector of claim 15.

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