Antibodies targeting human basal cell adhesion molecules

Abstract

Antibodies that specifically bind to human BCAM are described, as well as methods for preparing and using such antibodies. These antibodies could be used to treat cancers expressing BCAM.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 571,595, filed March 29, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to antibodies that specifically bind to human basal cell adhesion molecule (BCAM).

[0003] sequence list This application contains a sequence list, which has been electronically submitted in .xml format and is incorporated herein by reference in its entirety. The sequence list was created on February 13, 2024, named “3320.W01WO_Sequence Listing.xml”, and has a size of 134 kilobytes. Background Technology

[0004] High-grade serous ovarian cancer (HGSOC) is the most common and deadliest type of ovarian cancer. The vast majority of women diagnosed with it are already at an advanced stage. Current treatment options are limited to a combination of surgery and chemotherapy, and the cancer often recurs regardless of the treatment outcome. 70% of patients die within 5 years of being diagnosed with HGSOC.

[0005] Human basal cell adhesion molecule (BCAM) is a highly glycosylated transmembrane protein that acts as a receptor for laminin α-5. BCAM is highly expressed in a variety of tumors, but its expression is relatively low in most healthy tissues. High expression of BCAM is observed in 35%–40% of primary HGSOC tumors. However, it remains unclear whether laminin α-5 is the major ligand for BCAM in ovarian cancer. The nature of inhibitory effects targeting BCAM is also unclear.

[0006] Currently, the effective anti-BCAM antibody library for therapeutic use is limited. Therefore, there is still a need to identify antibodies that target BCAM and can be used to treat cancers such as HGSOC. Summary of the Invention

[0007] This disclosure relates to antibodies that specifically bind to human basal cell adhesion molecule (BCAM).

[0008] This article provides an antibody that specifically binds to human BCAM, wherein the antibody comprises a variable region and a constant region, wherein the variable region comprises a framework region and a complementary determining element for binding to human BCAM.

[0009] Specifically, this article provides an antibody that specifically binds to human BCAM, the antibody comprising: a) Heavy chain complementarity-determining regions (HCDRs) 1 containing the amino acid sequence DYYVH (SEQ ID NO: 2), HCDR 2 containing the amino acid sequence IINPFGGSATYAPKFQG (SEQ ID NO: 3), and HCDR 3 containing the amino acid sequence GIYGHFDY (SEQ ID NO: 4); and b) Light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence KSSQSVLSASNNQNYLA (SEQ ID NO: 5), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 6), and LCDR3 containing the amino acid sequence QQYYSSPYT (SEQ ID NO: 7).

[0010] This article also provides an antibody that specifically binds to human BCAM, the antibody comprising: a) HCDR1 containing the amino acid sequence SYAFS (SEQ ID NO: 8), HCDR2 containing the amino acid sequence GIIPFSGTTNYAQKFQG (SEQ ID NO: 9), and HCDR3 containing the amino acid sequence DPILSFWSGYYYYYGMDV (SEQ ID NO: 10); and b) LCDR1 containing the amino acid sequence RASQSISSWLA (SEQ ID NO: 11), LCDR2 containing the amino acid sequence DASNLET (SEQ ID NO: 12), and LCDR3 containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13).

[0011] This article also provides an antibody that specifically binds to human BCAM and competes with antibodies comprising: a) The variable (VH) region of the immunoglobulin heavy chain, which contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14), and The variable (VL) region of the immunoglobulin light chain contains an amino acid sequence. OR b) The variable (VH) region of the immunoglobulin heavy chain, which contains the amino acid sequence. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16), and The variable (VL) region of the immunoglobulin light chain contains an amino acid sequence. DIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

[0012] This article also provides an antibody that specifically binds to human BCAM, the antibody comprising: a) HCDR1 containing the amino acid sequence SYYMH (SEQ ID NO: 18), HCDR2 containing the amino acid sequence WMNPNSGDSGYAQKFQG (SEQ ID NO: 19), and HCDR3 containing the amino acid sequence TYSSSWIFDY (SEQ ID NO: 20); and b) LCDR1 containing the amino acid sequence KSSQSVLYSSNNKNYLA (SEQ ID NO: 21), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 22), and LCDR3 containing the amino acid sequence QQYYSIPIT (SEQ ID NO: 23).

[0013] This article also provides an antibody that specifically binds to human BCAM and competes with antibodies comprising: a) The variable (VH) region of the immunoglobulin heavy chain containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO: 24), and b) The variable (VL) region of the immunoglobulin light chain containing the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO:25).

[0014] Furthermore, this article provides an antibody-drug conjugate (ADC) comprising a component for killing tumor cells conjugated to an antibody that specifically binds to human BCAM via a linker, wherein the antibody comprises a variable region and a constant region, wherein the variable region comprises a framework region and a complementarity-determining component for binding to human BCAM. An ADC may comprise: a) Anti-BCAM antibody; b) Antitumor drugs; and c) Connectors that link anti-BCAM antibodies to anti-tumor drugs.

[0015] A dual epitope antibody is also provided, which comprises at least one antibody or ADC as described in any paragraph.

[0016] This article also provides a bispecific T-cell connector (BiTE) comprising an antibody component for binding human BCAM, an antibody component for binding CD3, and a connector component linking the antibody component for binding human BCAM to the antibody component for binding CD3. The BiTE may include: a) Anti-BCAM antibody; b) Anti-CD3 antibody; c) A linker connecting the anti-BCAM antibody to the anti-CD3 antibody; and optionally d) Components used to extend half-life (e.g., Fc region).

[0017] A pharmaceutical composition is also provided comprising an antibody, ADC, or BiTE as described herein, and one or more pharmaceutically acceptable excipients.

[0018] It also provides methods for treating cancer in subjects in need, which include administering a therapeutically effective amount of the antibody, ADC, or BiTE described herein to the subject.

[0019] definition A or an The articles “a” and “an” refer to one or more (i.e., at least one) grammatical object of the article. For example, “an element” means one or more elements.

[0020] Affinity The term "affinity" refers to the characteristic of the binding interaction between a binding component (e.g., an antigen-binding region, such as a variable domain containing light chain and heavy chain variable regions, and / or an Fc receptor-binding component, such as an Fc region) and a target (e.g., an antigen such as BCAM, or an Fc receptor (FcR) such as a neonatal FcR (FcRn)) and indicates the strength of the binding interaction. Affinity can be measured as the dissociation constant (K0). D The bonding component can have a high affinity for the target (e.g., K+ in the nanomolar or picomolar range). D The binding component can have a high affinity for the target at a first pH (e.g., pH 7-7.4) and a lower affinity for the target at a second pH (e.g., pH 4.5-5) (e.g., 1 / 10 or 1 / 100 of the former) (e.g., above about 100 nM, above about 1 µM, above about 10 µM, or above about 100 µM).

[0021] About or approximately: The term “approximately” or “about” applied to one or more values ​​of interest refers to a value similar to the reference value. Unless otherwise stated or obvious from the context, the term “approximately” or “about” may refer to a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above or below the reference value (e.g., ±5% or ±1%).

[0022] AntibodyThe term "antibody" refers to an antigen-binding polypeptide containing six complementarity-determining regions (CDRs). Exemplary antigen-binding polypeptides included in the term "antibody" include, but are not limited to, IgA antibodies, IgG antibodies, IgE antibodies, IgM antibodies, bispecific or multispecific antibodies (including bispecific T-cell conjugates (BiTEs) and biepitaxy antibodies), Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and single-chain variable fragments (scFv). Antibodies typically contain at least a heavy (H) chain variable region (abbreviated as VH herein) and at least a light (L) chain variable region (abbreviated as VL herein). Antibodies may contain two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, Fd, and Fv fragments), single-chain antibodies (e.g., single-chain Fv), and full-length antibodies, such as complete immunoglobulins (and their subtypes) of the IgA, IgG, IgE, IgD, and IgM types. Full-length antibodies typically contain constant regions. Full-length antibodies can be IgG, such as IgG1, IgG3, or IgG4. The light chains of immunoglobulins can be κ or λ types. An "antibody" can be an immunoglobulin molecule containing two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds, wherein the amino-terminal portion of each LC and HC contains a variable region of approximately 100-120 amino acids, which is primarily responsible for antigen recognition via the CDR contained therein. Scattered within the CDR are regions that are well-known and generally conserved within and between species (e.g., mice and humans); these are called frame regions (FRs).

[0023] Binding member "Binding component" is any molecule or part of a molecule (e.g., an antibody or an antigen-binding region or fragment of an antibody) that is capable of specifically binding to a target (e.g., a target of interest (e.g., an antigen such as BCAM)).

[0024] Antigen binding fragment or antigen binding region An antigen-binding fragment refers to the portion of an intact antibody that binds to an antigen (e.g., BCAM). An antigen-binding fragment may contain the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments. Antigen-binding fragments or regions may also contain single-chain antibodies, such as single-chain Fv (scFv).

[0025] Antibody member for binding :the term" Antibody member for binding"Antibodies that specifically bind to targets of interest (e.g., antigens such as BCAM) are described. For example, antibody components for binding human basal cell adhesion molecule (BCAM) are antibodies that specifically bind to human BCAM. Anti-BCAM antibodies are described herein and include those listed in Table 1, specifically MAb452, 477, and 929. Equivalent antibodies differ in their amino acid sequences but perform the same function of specifically binding BCAM through CDR-target interactions and achieve comparable results of inhibiting tumor growth alone or as an ADC or BiTE. Equivalent CDRs may also share at least 85% sequence identity with the disclosed antibodies and / or compete for binding to human BCAM, as determined by cell-based assays using fluorescence-activated cell sorting (FACS, see, for example, Example 4)."

[0026] Complementarity determining member for binding: the term" Complementarity determining member for binding The complementarity-determining regions (CDRs) of the antigen (BCAM) binding sites that constitute the antibodies are described. Specific CDRs are shown in Table 1. Equivalent CDRs differ in their amino acid sequences but perform the same function of specifically binding BCAM through CDR-target interactions, achieving comparable results of inhibiting tumor growth alone or as an ADC or BiTE. Equivalent CDRs may also share at least 85% sequence identity with the anti-BCAM antibodies of the present invention and / or compete for human BCAM binding, as determined by cell-based assays using fluorescence-activated cell sorting (FACS, see, for example, Example 4).

[0027] Complementarity determining region (CDR) The "complementarity-determining region" or "CDR" is an amino acid residue within the variable region, identified in this paper according to the definition given by Kabat et al. (see, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC). Other CDR boundary definitions may not strictly follow Kabat's approach but will still overlap with at least a portion of Kabat's CDR. For example, they may be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues do not significantly affect antigen binding.

[0028] Constant region The term "constant region" refers to a polypeptide that corresponds to or is derived from one or more constant immunoglobulin domains of an antibody. Constant regions may contain the following immunoglobulin domains: C H 1. Structural domain, hinge region, C H 2. Structural domain, C H3. Domains (derived from IgA, IgD, IgG, IgE, or IgM) and C H 4. Structural domains (derived from IgE or IgM). Typically, the constant region contains C... H 1. Structural domain, hinge region, C H 2. Structural domain, C H 3. Domains (derived from, for example, IgG1 or IgG4).

[0029] Epitope "Epitope" is a term known in the art and refers to a localized region of an antigen that an antibody can specifically bind to. An epitope can be, for example, a series of amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, an aggregation of two or more discontinuous regions or residues of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitope).

[0030] Fc region The term "Fc region" refers to a dimer of two "Fc polypeptides," each containing the constant region of the antibody, excluding the first constant region immunoglobulin domain. An "Fc region" may contain two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. An "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also contain part or all of the flexible hinge at the N-terminus of these domains. For IgG, an "Fc polypeptide" contains the lower portion of the hinge between the immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3 (Cγ3), and Cgamma1 (Cγ1) and Cγ2. Although the boundaries of Fc peptides can vary, human IgG heavy chain Fc peptides are generally defined as containing residues from T223 or C226 or P230 to their carboxyl terminus, where the numbering is based on the EU index in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, the Fc peptide contains the immunoglobulin domains Calpha2 (Cα2) and Calpha3 (Cα3) and the lower part of the hinge between Calpha1 (Cα1) and Cα2.

[0031] K a "K" a "" refers to the association rate of a specific binding component and a target to form a binding component / target complex.

[0032] K d "K"d "" refers to the dissociation rate of a specific binding component / target complex.

[0033] K D "K" D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a K was obtained and expressed as molar concentration (M). D The value can be determined using methods well-established in the art, such as by using surface plasmon resonance, for example using the BIACORE® system, or cell-based methods (using cells expressing human BCAM and flow cytometry, as described, for example, in Example 1). D The value can also be determined by biolayer interferometry using immersion-reading biosensors such as Sartorius Octet RH16 or Octet N1 (as described, for example, in Example 5).

[0034] Killing member or member for killing tumor cells :the term" Killing member "or" For killing tumor cells Member "Cell-killing agent" refers to any compound that can be used to kill cells, especially tumor cells. This includes, for example, cytotoxic drugs and radionuclides as described herein. The term "component for killing tumor cells" may be used interchangeably with the terms "antitumor drug" or "anticancer drug." Antitumor drugs and anticancer drugs include well-known classes of cytotoxic agents, such as tubulin inhibitors or DNA damaging agents, including, for example, DNA alkylating agents, topoisomerase inhibitors, and DNA cross-linking agents.

[0035] Member for extending half-life :the term" Member for extending half-life "Prolonged serum half-life" refers to well-known structural modifications that extend the serum half-life of an antibody in human serum. These include pegylation and fusion with human serum albumin (HSA) or HSA-binding components (e.g., anti-HSA antibodies). In some cases, the term can refer to the Fc region, such as an Fc region containing well-known structural modifications (e.g., amino acid substitutions that extend the serum half-life of the antibody). Such amino acid substitutions include, for example, M252Y / S254T / T256E (YTE mutant), L309D / Q311H / N434S (DHS mutant), V259I / V308F / M428L (IFL mutant), H433K / N434F (KF mutant), and M428L / N434S (LS mutant), which exhibit improved serum half-life relative to, for example, the native IgG1 Fc region.

[0036] Linking member or linker The term "connector" or "connector" refers to a well-known structure that links an antibody to two binding domains in a drug conjugate or a bispecific T-cell connector (BiTE). Exemplary connectors or connectors (including cleavable connectors) are described herein.

[0037] Specifically binds For the binding component and the target, "specific binding" or "specifically binding" refers to the preferential association of the binding component with the target, rather than with an entity that is not the target. Some degree of non-specific binding may occur between the binding component and the non-target. The binding component is considered to specifically bind to the target if the binding between the binding component and the target is greater than 2, 5, 10, or 100 times greater than the binding between the binding component and the non-target. Specific binding to human BCAM can be determined as described in Example 1.

[0038] Single chain variable fragment (scFv): The term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (e.g., mouse or human immunoglobulin) are covalently linked to form a VH::VL heterodimer. The heavy chain (VH) and light chain (VL) are linked directly or via a peptide-based linker (e.g., 10, 15, 20, 25 amino acids) that links the N-terminus of the VH to the C-terminus of the VL or vice versa. The linker is typically enriched with glycine for flexibility and serine or threonine for solubility. The linker may link the heavy chain variable region and light chain variable region of an antibody or its antigen-binding fragment. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010. Exemplary peptide-based linkers include GGGG (SEQ ID NO: 26) and GGGGS (SEQ ID NO: 27).

[0039] Subject The term "subject" refers to any subject who requires treatment. Typically, subjects are human subjects.

[0040] Therapeutically effective amount:The term "therapeutic effective amount" refers to the amount of a therapeutic molecule (e.g., the anti-BCAM antibody described herein) that imparts a therapeutic effect to the treated subject with a reasonable benefit / risk ratio applicable to any medical treatment. Therapeutic effect can be objective (i.e., measurable by some test or biomarker) or subjective (i.e., the subject gives an indication of the effect or feels the effect). Specifically, "therapeutic effective amount" refers to the amount of a therapeutic molecule or composition that effectively treats, improves, or prevents a particular disease or condition, or exhibits a detectable therapeutic or preventative effect, such as by improving disease-related symptoms, preventing or delaying the onset of the disease, and / or also reducing the severity or frequency of disease symptoms.

[0041] Treatment The term "treatment" (also known as "treat" or "treating") refers to any administration of a therapeutic molecule (e.g., the anti-BCAM antibody described herein) that partially or completely reduces, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or decreases the incidence of one or more symptoms or features of a particular disease, condition, and / or symptom. Such treatment may be directed to subjects who have not yet exhibited signs of the relevant disease, condition, and / or symptom and / or subjects who exhibit only early signs of the disease, condition, and / or symptom. More commonly, such treatment may be directed to subjects who exhibit one or more definitive signs of the relevant disease, condition, and / or symptom (e.g., cancer). Attached Figure Description

[0042] The accompanying drawings are for illustrative purposes only.

[0043] Figure 1 A schematic diagram of BCAM, MCAM, and a chimeric construct containing domains of both proteins is shown, which are used to narrow the location of the epitope to which the anti-BCAM antibody disclosed herein binds.

[0044] Figure 2 illustrates the binding of the anti-BCAM antibody exemplified by the present invention. Figure 1 A representative histogram of the capabilities of some BCAM / MCAM chimeric constructs is shown. Figure 2A A representative histogram obtained using anti-BCAM antibody 929 is shown. Figure 2B A representative histogram obtained using anti-BCAM antibody 477 is shown. Figure 2C A representative histogram obtained using anti-BCAM antibody 452 is shown.

[0045] Figure 3 shows representative curves depicting the ADCC activities of anti-BCAM antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 when administered to cells at various concentrations. Figure 3AAn exemplary anti-BCAM antibody is shown to target ADCC activity in OVSAHO cells. Figure 3B An exemplary anti-BCAM antibody is shown to target ADCC activity in OAW28 cells. Figure 3C An exemplary anti-BCAM antibody is shown to target ADCC activity in SKOV3-BCAM cells.

[0046] Figure 4 Line graphs showing the concentration-dependent effects of anti-BCAM antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 on the interaction between human BCAM and its ligand laminin α-5 are presented.

[0047] Figure 5 shows representative histograms illustrating the binding competition between anti-BCAM antibodies 228, 232, 236, 452, 477, 606, 929, and 937 and human BCAM. Figure 5A Representative histograms of experiments in which anti-BCAM antibodies were added to a pre-formed complex of antibody 452 and human BCAM are shown. Figure 5B The diagram shows a representative histogram of an experiment in which anti-BCAM antibody was added to a pre-formed complex of antibody 929 and human BCAM. Figure 5C The diagram shows a representative histogram of an experiment in which anti-BCAM antibody was added to a pre-formed complex of antibody 477 and human BCAM.

[0048] Figure 6 shows a representative histogram illustrating how the presence of point mutations in BCAM affects the binding of the anti-BCAM antibody of the present invention. Figure 6A A representative histogram depicting the effect of point mutations on the ability of antibody 477 to bind to human BCAM is shown. Figure 6B A representative histogram depicting the effect of point mutations on the ability of antibody 452 to bind to human BCAM is shown. Figure 6C A representative histogram depicting the effect of point mutations on the ability of antibody 929 to bind to human BCAM is shown.

[0049] Figure 7A The amino acid sequences of human, cynomolgus monkey, and mouse BCAM are shown in the diagram. The sequence of human BCAM used is residues 1 to 516 of SEQ ID NO: 1. The sequence of cynomolgus monkey BCAM used is SEQ ID NO: 28, which corresponds to residues 32 to 547 of UniProtKB ID: A0A2K5TW87_MACFA. The sequence of mouse BCAM used is SEQ ID NO: 29, which corresponds to residues 26 to 541 of UniProtKB ID: Q9R069.

[0050] Figure 7BA representative histogram depicting the effect of point mutations in human BCAM on the ability of antibody 929 to bind to human BCAM is shown.

[0051] Figure 7C A representative histogram depicting the effect of point mutations in human BCAM on the ability of antibody 477 to bind to human BCAM is shown.

[0052] Figure 8 Representative histograms are shown illustrating the effects of various polymorphisms of human BCAM on the binding of anti-BCAM antibodies 452, 477, and 929.

[0053] Figure 9 shows a representative biolayer interferometry (BLI) curve of the anti-BCAM antibody. Figure 9A A representative BLI curve for anti-BCAM antibody 452 is shown. Figure 9B A representative BLI curve for anti-BCAM antibody 477 is shown. Figure 9C A representative BLI curve for anti-BCAM antibody 929 is shown.

[0054] Figure 10A A schematic diagram of a bispecific T-cell adaptor (BiTE) construct is shown, which includes an anti-BCAM binding component (i.e., a single-stranded variable fragment (scFv) that specifically binds to BCAM) linked to an anti-CD3 binding component (i.e., a scFv that specifically binds to CD3). Figure 10B It shows Figure 10A A schematic diagram of the BiTE construct, which also includes components for extending the half-life (i.e., human IgG1-Fc with the LALA-PG mutation).

[0055] Figure 11A Representative graphs illustrating the effects of the anti-BCAM BiTE construct on T cell activation at various concentrations are shown. Figure 11B A representative graph illustrating the percentage of activated T cells after incubating donor PBMCs with a 477-based BiTE construct is shown. The normalized percentage of target cells lysed due to T cell activation after incubating donor PBMCs with a 477-based BiTE construct is shown accordingly. Figure 11C middle.

[0056] Figure 12A A representative plot demonstrating the activation of T cells incubated in the presence of wild-type (WT) SKOV3 target cells by the 477-based anti-BCAM BiTE-Fc construct described herein is shown. The percentage of target SKOV3 WT cells lysed due to T cell activation with this BCAM BiTE-Fc construct is shown accordingly. Figure 12B middle. Figure 12CA representative plot demonstrating the activation of T cells incubated in SKOV3 cells modified to express recombinant human BCAM by the 477-based anti-BCAM BiTE-Fc construct described herein is shown. The percentage of modified SKOV3 cells lysed due to T cell activation with this BCAM BiTE-Fc construct is shown accordingly. Figure 12D middle.

[0057] Figure 13A A representative plot demonstrating the activation of T cells incubated in the presence of OVSAHO target cells by the 477-based anti-BCAM BiTE-Fc construct described herein is shown. The percentage of OVSAHO cells lysed due to T cell activation with the anti-BCAM BiTE-Fc construct is shown accordingly. Figure 13B middle.

[0058] Figure 14 A representative graph is shown demonstrating that 477-based anti-BCAM BiTE-Fc can effectively prevent the growth of tumors established in mice using SKOV3-BCAM cells.

[0059] Figure 15A Representative graphs demonstrating the internalization of each of the anti-BCAM antibodies 452, 477, and 929 into BCAM-expressing cells are shown.

[0060] Figure 15B Representative confocal microscopy images demonstrating that OVSAHO cells can internalize antibody-drug conjugates (ADCs) containing anti-BCAM antibodies 452, 477, or 929 conjugated to delutecan (DXd) are shown. ADCs internalized into the low-pH environment within the cell (i.e., lysosomes and endosomes) are shown as white dots. Arrows point to the internalized ADCs.

[0061] Figure 16 Representative graphs demonstrating the ability of the ADC containing the anti-BCAM antibody of the present invention to bind human BCAM at a drug-antibody ratio of 4 (DAR4) or 8 (DAR8) are shown. The drug is delutecan (DXd).

[0062] Figure 17 Representative graphs are shown demonstrating that ADCs based on anti-BCAM antibodies 477, 452, or 929 at picomolar concentrations (drug-antibody ratio of 8) (DAR8) can reduce the viability of OVCAR8 (OV8) cells.

[0063] Figure 18 shows a representative graph demonstrating that ADCs containing the anti-BCAM antibody of the present invention at picomolar concentrations can equally reduce the viability of OVCAR8 (OV8) cells at antibody-drug ratios of 4 (DAR4) or 8 (DAR8). Figure 18A Cell viability after treatment with an ADC based on anti-BCAM antibody 929 is shown. Figure 18B Cell viability after treatment with an ADC based on anti-BCAM antibody 452 is shown. Figure 18C Cell viability after treatment with an ADC based on anti-BCAM antibody 477 is shown.

[0064] Figure 19 shows representative graphs demonstrating reduced cell viability in various cancer cell lines after administration of the exemplary anti-BCAM ADC described herein. The anti-BCAM antibody is Antibody 477, and the drug is delutecan (DXd). Figure 19A This showed a decrease in OVSAHO cell viability. Figure 19B This showed a decrease in Kuramochchi cell viability. Figure 19C This showed a decrease in OVCAR3 cell viability. Figure 19D This study showed a decrease in the viability of OVCAR8 (OV8) cells. Figure 19E This showed a decrease in the viability of MFE-280 cells.

[0065] Figure 20 A representative graph is shown demonstrating the sustained reduction in tumor volume in NSG mice treated with an anti-BCAM antibody 477-based ADC containing Dxd in DAR4, compared to treatment with an anti-RSV ADC (containing Dxd at an antibody-drug ratio of 4) and mediator only (negative control).

[0066] Figure 21 A representative ADC containing a dual-epitope anti-BCAM antibody is shown. This dual-epitope anti-BCAM ADC comprises (1) a first full-length anti-BCAM antibody containing the IgG Fc region, and (2) a second anti-BCAM single-chain variable fragment (scFv), wherein two copies are fused to the C-terminus of the IgG Fc region. The full-length anti-BCAM antibody is labeled “α-BCAM IgG (1)” and shown in black and light gray. The scFv is labeled “α-BCAM scFv (2)” and shown in dark gray and white. The two drug portions attached to the IgG Fc region are indicated by vertical striped ellipses.

[0067] Figure 22 shows a representative plot demonstrating the phenomenon of picomolar concentration. Figure 21The dual-epitope anti-BCAM ADC, as shown and containing MMAE as a drug, can reduce the viability of OVCAR3 cells at an antibody-drug ratio of 2 (DAR2). Figure 22A Cell viability is shown after treatment with a dual epitope anti-BCAM ADC (“929-477scFv ADC”) containing 929-based IgG1 and 477-based scFv. Figure 22B Cell viability is shown after treatment with a dual epitope anti-BCAMADC (“452-477scFv ADC”) containing IgG1 based on 452 and scFv based on 477. Figure 22C Cell viability after treatment with a biepisode anti-BCAM ADC (“452-929scFv ADC”) comprising 452-based IgG1 and 929-based scFv is shown. ADCs of the component antibodies of the biepisode constructs tested are included as controls. The control ADC is formatted as a full-length IgG antibody. Figure 21 The nomenclature is used to indicate whether the control ADC forms α-BCAM IgG (1) or α-BCAM scFv (2). IgG containing MMAE (“IgG-oYo”) is included as a negative control. Detailed Implementation

[0068] This article provides antibodies that specifically bind to BCAM, specifically human BCAM. It also provides methods for treating cancer using the anti-BCAM antibodies of this invention.

[0069] Basal cell adhesion molecules (BCAM) BCAM, also known in the art as Lutheran antigen and CD239, is a glycoprotein that has been described as a receptor for laminin α-5.

[0070] BCAM contains five immunoglobulin superfamily extracellular domains, a single transmembrane domain, and a short C-terminal cytoplasmic tail. The five extracellular domains, from N-terminus to C-terminus, comprise V1, V2, C1, C2, and C3 (referred to accordingly as D1, D2, D3, D4, and D5). Laminin α-5 binding is expected to involve residues in the V2 and / or C1 domains, as well as residues in the region connecting these two domains. Specifically, residues E309, D310, and D312 of the amino acid sequence of human BCAM, as shown in SEQ ID NO: 1, have been shown to be important for laminin α-5 binding (Mankelow et al., 2007). Blood 110(9):3398-3406).

[0071] Two transcript variants of human BCAM have been identified in this art (the sequences of which have been previously deposited with NCBI reference numbers NM_005581.5 and NM_001013257.2). The difference between these two transcript variants is the length of the C-terminal cytoplasmic tail. The shorter of the two isotypes lacks C-terminal residues 558 to 597 of SEQ ID NO: 1. The N-terminal region of each sequence contains a cleaved signal peptide, such that BCAM on the cell surface typically does not contain an N-terminal signal peptide.

[0072] The amino acid sequence of human BCAM is shown below. This sequence does not contain the first 31 amino acids containing the N-terminal signal peptide sequence. The residue numbering used herein begins with the first residue E as shown below. SEQ ID NO: 1 corresponds to residues 32 to 628 of UniProtKB ID: P50895.

[0073] (SEQ ID NO: 1).

[0074] Anti-BCAM antibody The anti-BCAM antibodies of the present invention are capable of specifically binding to BCAM, specifically to BCAM. Typically, such antibodies are provided as isolated antibodies, specifically isolated monoclonal antibodies (“MAb”). MAb is an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone). The amino acid sequences of exemplary anti-BCAM antibodies of this disclosure, along with their CDR and variable chain sequences, are shown in Table 1.

[0075] Table 1. Anti-BCAM antibody amino acid sequences

[0076] The anti-BCAM antibodies of the present invention have been selected, along with other parameters, due to their high affinity and specificity for human BCAM and their ability to bind to human BCAM polymorphisms. For example, MAb 228, 232, 452, 477, 606, 929, and 937 can each bind to at least eight different human BCAM polymorphisms. Therefore, the anti-BCAM antibodies can bind to at least nine different human BCAM polymorphisms, such as MAb 228, 232, 452, 477, 606, and 929. For example, the anti-BCAM antibodies can bind to nine or more of Lu21, Lu20, Lu17, Lu16, Lu13, Lu12, Lu8, Lu6, Lu5, Lu4, and Lu2. Other features of the anti-BCAM antibodies of the present invention may include the ability of the antibody to be efficiently internalized into cells after binding to human BCAM and the absence of potential sequence risk factors.

[0077] For example, the anti-BCAM antibody of the present invention can satisfy at least the first three requirements listed in Table 2. In some cases, the anti-BCAM antibody of the present invention can satisfy at least the first three requirements and the last requirement listed in Table 2. Alternatively, the anti-BCAM antibody of the present invention can satisfy all the requirements listed in Table 2.

[0078] Table 2: Binding characteristics Parameters Requirements Binding to BCAM knockout cells <2.5% binding Cross-reactivity with homologs RAGE, MCAM, and ALCAM <5% binding K D (as determined by a cell-based assay) <5 nM K D (as determined by BLI) <1 nM K D (as determined by ELISA) <6 nM Number of BCAM polymorphisms bound ≥9 Antibodies can be prepared, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR or specificity-determining residues, SDR, transplantation), or a combination of such technologies or other technologies known in the art. The use of hybridoma technology typically involves immunizing an animal (e.g., a mouse) with the extracellular portion of human BCAM. A variety of well-known methods and tools can be used to prepare and purify the anti-BCAM antibodies of the present invention, including vectors such as plasmids, viruses, or other mediators for polynucleotide insertion or expression, and hosts such as microorganisms, yeast, insects, and mammalian organisms (see, for example, Process Scale Purification of Antibodies, ed. Uwe Gottschalk, 2nd edition, 2017).

[0079] Modulation of binding to laminin alpha-5 The anti-BCAM antibodies of the present invention can modulate the binding of human BCAM to laminin α-5 (e.g., MAb 232, 452, 477, 929, and 937). For example, the anti-BCAM antibodies of the present invention (e.g., MAb 232, 477, 929, and 937) can reduce, weaken, or inhibit the binding of BCAM to laminin α-5. Alternatively, the anti-BCAM antibody of the present invention (e.g., MAb 452) can increase the binding of BCAM to laminin α-5. As demonstrated herein, both such agonist and antagonist antibodies can specifically bind to human BCAM on cancer cells and reduce the viability of BCAM-expressing cancer cells. In some cases, the anti-BCAM antibodies of the present invention may not modulate the binding of human BCAM to laminin α-5 (e.g., MAb 228 and 606).

[0080] Anti-BCAM antibodies may contain an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is approximately 85% or more identical (e.g., approximately 90% or 95% identical) to or the same as the following sequences: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14); QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16); QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO: 24); OR EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSS (SEQ ID NO: 72).

[0081] Anti-BCAM antibodies may contain an immunoglobulin light chain variable (VL) region containing an amino acid sequence that is approximately 85% or more identical (e.g., approximately 90%, approximately 95%) or the same as the following sequences: DIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 15); DIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17); DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25); OR DIQMTQSPSSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIK (SEQ ID NO: 73).

[0082] Anti-BCAM antibodies may contain an immunoglobulin heavy chain variable (VH) region containing approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14), and The immunoglobulin light chain variable (VL) region contains about 85%, about 90%, about 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as DIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 15).

[0083] Anti-BCAM antibodies may contain an immunoglobulin heavy chain variable (VH) region containing approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16), and The immunoglobulin light chain variable (VL) region contains approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

[0084] Anti-BCAM antibodies may contain an immunoglobulin heavy chain variable (VH) region containing approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO: 24), and It contains an immunoglobulin light chain variable (VL) region containing about 85%, about 90%, about 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25).

[0085] Anti-BCAM antibodies may contain an immunoglobulin heavy chain variable (VH) region containing approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGDTNYAQEFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARKRDGGGPFDYWGQGTLVTVSS (SEQ ID NO: 46), and The immunoglobulin light chain variable (VL) region contains approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as DIVMTQSPDSLAVSLGERATINCKSSQSVLSRSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVDIKR (SEQ ID NO: 47).

[0086] Anti-BCAM antibodies may contain an immunoglobulin heavy chain variable (VH) region containing approximately 85%, approximately 90%, approximately 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSS (SEQ ID NO: 72), and The immunoglobulin light chain variable (VL) region contains about 85%, about 90%, about 95% or more (e.g., at least 85%) of the same or identical amino acid sequence as DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIK (SEQ ID NO: 73).

[0087] Anti-BCAM antibodies can have dissociation constants (Kc) in the nanomolar range. D Alternatively, anti-BCAM antibodies can have a Kc in the picomolar range. D For example, the K of antibodies DIt can be about 100 nM or less, for example, about 0.001 nM to about 100 nM, or about 0.01 nM to about 10 nM. Alternatively, the K of the antibody... D It can be about 10 nM or smaller, for example, about 0.01 nM to about 10 nM.

[0088] K D The K of the anti-BCAM antibody can be determined using cell-based assays, such as those shown in Example 1. D It can be about 10 nM or less, for example, about 0.01 nM to about 10 nM, as determined using cell-based assays. K of anti-BCAM antibodies D It can be about 5 nM or less, for example, about 0.01 nM to about 5 nM, as determined using cell-based assays. K of anti-BCAM antibodies D It can be approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, or approximately 1 nM, as determined using a cell-based assay. K of the anti-BCAM antibody D It can be about 1 nM or less, for example, about 0.1 nM to about 1 nM, as determined using cell-based assays. For example, the K of anti-BCAM antibodies D It can be about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM or about 0.1 nM, as determined using a cell-based assay.

[0089] K D This can be determined using an enzyme-linked immunosorbent assay (ELISA), for example, as shown in Example 1. Therefore, the K of the anti-BCAM antibody... D It can be less than 10 nM, typically less than 6 nM, for example, from about 0.01 nM to about 5 nM, as determined by ELISA. K of anti-BCAM antibody D It can be approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, or approximately 1 nM, as determined by ELISA. The K of the anti-BCAM antibody... D It can be about 1 nM or less, for example, about 0.1 nM to about 1 nM, as determined by ELISA. For example, the K of anti-BCAM antibodies. D It can be approximately 0.9 nM, approximately 0.8 nM, approximately 0.7 nM, approximately 0.6 nM, approximately 0.5 nM, approximately 0.4 nM, approximately 0.3 nM, approximately 0.2 nM or approximately 0.1 nM, as determined by ELISA.

[0090] K DBiological layer interferometry (BLI) can be used to determine, for example, as shown in Example 5. Therefore, the K of the anti-BCAM antibody... D It can be about 1 nM or less, for example, about 0.01 nM to about 1 nM, as determined by BLI. K of anti-BCAM antibodies D It can be approximately 0.9 nM, approximately 0.8 nM, approximately 0.7 nM, approximately 0.6 nM, approximately 0.5 nM, approximately 0.4 nM, approximately 0.3 nM, approximately 0.2 nM, or approximately 0.1 nM, as determined by BLI. The K of the anti-BCAM antibody... D It can be about 0.1 nM or less, for example, about 0.01 nM to about 0.1 nM, as determined by BLI. For example, the K of anti-BCAM antibodies. D It can be about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.07 nM, about 0.06 nM, or about 0.05 nM, about 0.04 nM, about 0.03 nM, about 0.02 nM, or about 0.01 nM, as determined by BLI.

[0091] Antagonist antibody The anti-BCAM antibody of the present invention can inhibit the binding of BCAM to laminin α-5. This antibody can specifically bind to epitopes (e.g., discontinuous epitopes) comprising L9, K16, S17, I19, T25, G26, T27, H28, D29, T38, R40, S41, P45, A50, M52, L57, Q58, V59, M61, D63, T64, R65, S68, Q72, L73, Q76, L81, E83, A84, E89, R97, A104, A108, R109, L110, N111, K One or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acids of 115, A118, E132, D133, S134, N142, R144, A149, K151, V165, M167, P169, E170, M173, S175, E180, L185, R196, S203, S211, E214, R216, Q237, P223, and H226.

[0092] An epitope (e.g., a discontinuous epitope) may contain one or more (e.g., two, three, four, five, or six) amino acids selected from human BCAM, namely K16, R40, S68, E83, K115, N142, R144, E180, and P223. For example, an epitope (e.g., a discontinuous epitope) may contain the human BCAM amino acid R40. Alternatively, an epitope (e.g., a discontinuous epitope) may contain the human BCAM amino acid E180. Alternatively, an epitope (e.g., a discontinuous epitope) may contain the human BCAM amino acid P223.

[0093] The binding of an anti-BCAM antibody to an epitope (e.g., a discontinuous epitope) as defined above can be determined by contacting the antibody with a human BCAM variant in which one or more residues at positions 16, 40, 68, 83, 115, 142, 144, 180, and 223 have been substituted with a different amino acid, such as alanine (e.g., E180A). The human BCAM variant may contain one or more substitutions selected from K16E, R40E, S68R, E83K, K115D, N142S, R144N, and P223H. For example, an anti-BCAM antibody capable of inhibiting the binding of BCAM to laminin α-5 may not bind to a human BCAM variant in which the residue at position 180 of SEQ ID NO: 1 is substituted with alanine. Similarly, an anti-BCAM antibody capable of inhibiting the binding of BCAM to laminin α-5 may not bind to a human BCAM variant in which the residue at position 223 of SEQ ID NO: 1 is substituted with histidine. Anti-BCAM antibodies capable of inhibiting the binding of BCAM to laminin α-5 may not bind to: (i) human BCAM variants in which the residue at position 180 of SEQ ID NO: 1 is replaced by alanine, and (ii) human BCAM variants in which the residue at position 223 of SEQ ID NO: 1 is replaced by histidine. Anti-BCAM antibodies capable of inhibiting the binding of BCAM to laminin α-5 may not bind to human BCAM variants in which the residue at position 40 of SEQ ID NO: 1 is replaced by glutamic acid.

[0094] If binding to a human BCAM variant is reduced by at least 80% (e.g., 90% or 95%) relative to wild-type human BCAM, then the anti-BCAM antibody is determined to bind to an epitope as defined above. For example, binding can be determined using cells expressing human BCAM or a human BCAM variant (e.g., 293 cells) by flow cytometry (e.g., as described in Example 5). This document provides anti-BCAM antibodies (e.g., monoclonal antibodies) comprising a complementarity-determining region capable of specifically binding to such epitopes.

[0095] Antibodies capable of inhibiting the binding of BCAM to laminin α-5 may contain a heavy chain complementarity-determining region (HCDR) 1 containing the amino acid sequence DYYVH (SEQ ID NO: 2), an HCDR 2 containing the amino acid sequence IINPFGGSATYAPKFQG (SEQ ID NO: 3), an HCDR 3 containing the amino acid sequence GIYGHFDY (SEQ ID NO: 4), a light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence KSSQSVLSASNNQNYLA (SEQ ID NO: 5), an LCDR 2 containing the amino acid sequence WASTRES (SEQ ID NO: 6), and an LCDR 3 containing the amino acid sequence QQYYSSPYT (SEQ ID NO: 7).

[0096] The antibody may contain an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14), and The immunoglobulin light chain variable (VL) region contains an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to that of DIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 15).

[0097] Antibodies capable of inhibiting the binding of BCAM to laminin α-5 may comprise a single-stranded variable fragment (scFv) containing a sequence having at least about 85% (e.g., 90% or 95%) identity with the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGS GGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 78).

[0098] Antibodies containing such CDRs or heavy chain variable regions and light chain variable regions may not bind to human BCAM variants in which the residue at position 180 of SEQ ID NO: 1 is replaced by alanine and / or human BCAM variants in which the residue at position 223 of SEQ ID NO: 1 is replaced by histidine.

[0099] antibody K D It can be approximately 10 nM or smaller, for example, approximately 0.01 nM to approximately 10 nM. The K of the antibody D It can be about 1 nM or less, about 0.01 nM to about 1 nM, for example about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.4 nM, about 0.2 nM or about 0.1 nM.

[0100] antibody K D It can be approximately 0.7 nM, as determined using cell-based assays. The K of the antibody... D It can be approximately 0.5 nM, as determined using BLI. The K of the antibody... D It can be approximately 0.4 nM, as determined using ELISA.

[0101] The antibody may contain an immunoglobulin heavy chain variable (VH) region with the same amino acid sequence as QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14), and It contains the variable (VL) region of the immunoglobulin light chain, which has the same amino acid sequence as DIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 15).

[0102] Antibodies can contain single-chain variable fragments (scFvs) with sequences identical to the following: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGS GGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 78).

[0103] Alternatively, antibodies capable of inhibiting the binding of BCAM to laminin α-5 may comprise HCDR1 containing the amino acid sequence SYAFS (SEQ ID NO: 8), HCDR2 containing the amino acid sequence GIIPFSGTTNYAQKFQG (SEQ ID NO: 9), and HCDR3 containing the amino acid sequence DPILSFWSGYYYYYGMDV (SEQ ID NO: 10); as well as LCDR1 containing the amino acid sequence RASQSISSWLA (SEQ ID NO: 11), LCDR2 containing the amino acid sequence DASNLET (SEQ ID NO: 12), and LCDR3 containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13).

[0104] The antibody may contain an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16), and The immunoglobulin light chain variable (VL) region contains an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to that of DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

[0105] Antibodies capable of inhibiting the binding of BCAM to laminin α-5 may comprise a single-stranded variable fragment (scFv) containing a sequence having at least about 85% (e.g., 90% or 95%) identity with the following sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 79).

[0106] Antibodies containing such CDR or VH and VL regions may not bind to variants of human BCAM in which the residue at position 40 of SEQ ID NO: 1 is replaced by glutamic acid.

[0107] antibody K D It can be approximately 10 nM or smaller, for example, approximately 0.01 nM to approximately 10 nM. The K of the antibody DIt can be about 2 nM or smaller, for example, about 0.01 nM to about 2 nM, such as about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.4 nM, about 0.2 nM, or about 0.1 nM. The K of the antibody D It can be about 0.1 nM or less, such as about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM, about 0.04 nM, about 0.03 nM, about 0.02 nM or about 0.01 nM.

[0108] antibody K D It can be approximately 1.2 nM, as determined using cell-based assays. The K of the antibody... D It can be approximately 0.06 nM, as determined using BLI. The K of the antibody... D It can be approximately 0.7 nM, as determined using ELISA.

[0109] The antibody may contain an immunoglobulin heavy chain variable (VH) region containing the same amino acid sequence as QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16), and It contains the variable (VL) region of the immunoglobulin light chain, which has the same amino acids as DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

[0110] Antibodies can contain single-chain variable fragments (scFvs) with sequences identical to the following: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 79).

[0111] Alternatively, an antibody capable of inhibiting the binding of BCAM to laminin α-5 may comprise a heavy chain complementarity-determining region (HCDR) 1 containing the amino acid sequence SYYMH (SEQ ID NO: 40), an HCDR 2 containing the amino acid sequence WINPNSGDTNYAQEFQG (SEQ ID NO: 41), an HCDR 3 containing the amino acid sequence KRDGGGPFDY (SEQ ID NO: 42), a light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence KSSQSVLSRSNNKNYLA (SEQ ID NO: 43), an LCDR 2 containing the amino acid sequence WASTRES (SEQ ID NO: 44), and an LCDR 3 containing the amino acid sequence QQYYSTPFT (SEQ ID NO: 45).

[0112] The antibody may contain an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGDTNYAQEFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARKRDGGGPFDYWGQGTLVTVSS (SEQ ID NO: 46), and The immunoglobulin light chain variable (VL) region contains an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to that of DIVMTQSPDSLAVSLGERATINCKSSQSVLSRSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVDIKR (SEQ ID NO: 47).

[0113] Antibodies capable of inhibiting the binding of BCAM to laminin α-5 comprise a single-stranded variable fragment (scFv) containing a sequence having at least about 85% (e.g., 90% or 95%) identity with the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGDTNYAQEFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARKRDGGGPFDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSRSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVDIK (SEQ ID NO: 80).

[0114] antibody K D It can be approximately 10 nM or smaller, for example, approximately 0.01 nM to approximately 10 nM. The K of the antibody D It can be about 5 nM or smaller, for example, about 0.1 nM to about 5 nM. The K of the antibody D It can be approximately 4 nM, approximately 3 nM, approximately 2 nM, or approximately 1 nM. The K of the antibody... D It can be about 1 nM or smaller, for example about 0.1 nM to about 1 nM, such as about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.4 nM, about 0.2 nM or about 0.1 nM.

[0115] antibody K D It can be approximately 2 nM, as determined using cell-based assays. The K of the antibody... D It can be approximately 0.6 nM, as determined using ELISA.

[0116] Alternatively, antibodies capable of inhibiting the binding of BCAM to laminin α-5 may comprise HCDR1 containing the amino acid sequence DYAMH (SEQ ID NO: 66), HCDR2 containing the amino acid sequence VISYDGSHMSYADSVKG (SEQ ID NO: 67), and HCDR3 containing the amino acid sequence GGQLVWHPPDY (SEQ ID NO: 68); as well as LCDR1 containing the amino acid sequence RASQGISSYLA (SEQ ID NO: 69), LCDR2 containing the amino acid sequence AASYLQS (SEQ ID NO: 70), and LCDR3 containing the amino acid sequence QQSYRTPFT (SEQ ID NO: 71).

[0117] The antibody may contain an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSS (SEQ ID NO: 72), and The immunoglobulin light chain variable (VL) region contains an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to that of DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIK (SEQ ID NO: 73).

[0118] Antibodies capable of inhibiting the binding of BCAM to laminin α-5 may comprise a single-stranded variable fragment (scFv) containing a sequence having at least about 85% (e.g., 90% or 95%) identity with the following sequence: EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIK (SEQ ID NO: 81).

[0119] antibody K D It can be approximately 10 nM or smaller, for example, approximately 0.01 nM to approximately 10 nM. The K of the antibody D It can be about 5 nM or smaller, for example, about 0.1 nM to about 5 nM. The K of the antibody D It can be approximately 4 nM, approximately 3 nM, approximately 2 nM, or approximately 1 nM.

[0120] antibody K D It can be approximately 1.8 nM, as determined using cell-based assays.

[0121] The antibody may contain an immunoglobulin heavy chain variable (VH) region with the same amino acid sequence as EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSS (SEQ ID NO: 72), and It contains the variable (VL) region of the immunoglobulin light chain, which has the same amino acids as DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIK (SEQ ID NO: 73).

[0122] Antibodies can contain single-chain variable fragments (scFvs) with sequences identical to the following: EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIK (SEQ ID NO: 81).

[0123] In some cases, optimized (e.g., affinity-matured) variant anti-BCAM antibodies are provided, which may contain one or more substitutions in one or more CDR or framework residues without negatively impacting the binding properties of the parent antibody as defined in the preceding paragraphs (e.g., without reduction or alteration). Alternatively or additionally, variant anti-BCAM antibodies may be modified to remove one or more sequence risk factors.

[0124] Agonist antibody It also provides an anti-BCAM antibody that can increase the binding of BCAM to laminin α-5.

[0125] This antibody may contain HCDR1 containing the amino acid sequence SYYMH (SEQ ID NO: 18), HCDR2 containing the amino acid sequence WMNPNSGDSGYAQKFQG (SEQ ID NO: 19), HCDR3 containing the amino acid sequence TYSSSWIFDY (SEQ ID NO: 20), LCDR1 containing the amino acid sequence KSSQSVLYSSNNKNYLA (SEQ ID NO: 21), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 22), and LCDR3 containing the amino acid sequence QQYYSIPIT (SEQ ID NO: 23).

[0126] The antibody may contain an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO: 24), and The immunoglobulin light chain variable (VL) region contains an amino acid sequence that is at least 85% identical (e.g., at least 95% identical) to that of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25).

[0127] Antibodies that can increase the binding of BCAM to laminin α-5 may contain a single-stranded variable fragment (scFv) having at least about 85% (e.g., 90% or 95%) identity with the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 82).

[0128] antibody K D It can be about 10 nM or smaller, for example, about 0.01 nM to about 10 nM. In some embodiments, the K of the antibody... D Less than 6 nM, for example, from about 0.01 nM to about 5 nM. The K of the antibody. D It can be approximately 5 nM, approximately 4 nM, approximately 3 nM, approximately 2 nM, or approximately 1 nM. The K of the antibody... DIt can be about 1 nM or smaller, for example about 0.1 nM to about 1 nM, such as about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.4 nM, about 0.2 nM or about 0.1 nM.

[0129] antibody K D It can be approximately 0.3 nM, as determined using cell-based assays. The K of the antibody... D It can be approximately 5 nM, as determined using an ELISA. The K of the antibody... D It can be approximately 0.8 nM, as determined using BLI.

[0130] The antibody may contain an immunoglobulin heavy chain variable (VH) region with the same amino acid sequence as QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO: 24), and It contains the variable (VL) region of the immunoglobulin light chain, which has the same amino acid sequence as DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25).

[0131] Antibodies can contain single-chain variable fragments (scFvs) with sequences identical to the following: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 82).

[0132] In some cases, optimized (e.g., affinity-matured) variant anti-BCAM antibodies may be provided, which contain one or more substitutions in one or more CDR or framework residues without negatively impacting the binding properties of the parent antibody (e.g., without reduction or alteration). Alternatively or additionally, variant anti-BCAM antibodies may be modified to remove one or more sequence risk factors.

[0133] Other antibodies Other antibodies (e.g., other human antibodies) having complementary determinant components for binding human BCAM that are equivalent to the antibodies exemplified herein (e.g., Mab 452, 477, and 929) can be prepared or identified by well-known methods, such as immunization of transgenic animals and isolation of hybridomas, and screening of phage or yeast display libraries (see, for example, Monoclonal Antibodies: Methods and Protocols, edited by Vincent Ossipow and Nicolas Fischer, 2nd ed., 2014). Additionally, variant antibodies can be provided that contain one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) substitutions in one or more CDR or framework residues without negatively impacting (e.g., without degrading) the binding properties of the parent antibody.

[0134] Antibody-binding components or complementarity-determining components (e.g., antibodies) for binding human BCAM that are equivalent to the anti-BCAM antibody of the present invention differ in their amino acid sequences but perform the same function of binding the target via CDR-target interaction and act in the same manner (e.g., as an agonist or antagonist) and / or achieve comparable results (such as inhibition of tumor growth). For example, the complementarity-determining component for binding human BCAM is an antibody with 85% identity to the anti-BCAM antibody disclosed herein and inhibits tumor growth alone or as an ADC or BiTE.

[0135] Complementary determinant components for binding human BCAM can function by binding to epitopes that are identical to or overlap with the epitopes on human BCAM bound by the antibodies described herein. Antibodies having such complementary determinant components for binding human BCAM can be identified by their ability to compete with anti-BCAM antibodies (e.g., MAb 477 and 929) described herein for binding human BCAM. This binding competition can be determined by assays in which the anti-BCAM antibodies of the present invention provided herein prevent, inhibit, or reduce the specific binding of the test antibody, and vice versa, as demonstrated, for example, in Example 4.

[0136] If the binding of the anti-BCAM antibody of the present invention to human BCAM is reduced by at least 10% or more (e.g., 20% or more, 30% or more, or 40% or more) compared to the binding of the anti-BCAM antibody of the present invention to human BCAM alone, then the test antibody is determined to compete with the anti-BCAM antibody of the present invention. Specifically, if the binding of the anti-BCAM antibody of the present invention to human BCAM is reduced by at least 50% or more (e.g., 60% or more, 70% or more, or 80% or more) compared to the binding of the anti-BCAM antibody of the present invention to human BCAM alone, then the test antibody is determined to compete with the anti-BCAM antibody of the present invention.

[0137] Antibodies that bind to the same epitope as the anti-BCAM antibody of the present invention compete with said antibody for binding to human BCAM. Similarly, as demonstrated in the examples provided herein, antibodies that bind to epitopes that overlap with or are adjacent to the epitope bound by the anti-BCAM antibody of the present invention compete with said antibody for binding to human BCAM. Typically, overlapping epitopes contain at least one residue identical to at least one residue of the epitope of interest.

[0138] Typically, adjacent epitopes do not share any residues with the epitope of interest, but the binding of another antibody to an adjacent epitope reduces the binding of the anti-BCAM antibody of the present invention to human BCAM by at least 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more) compared to the binding of the anti-BCAM antibody of the present invention alone to human BCAM.

[0139] Many types of competitive binding assays can be used to determine whether the test antibody competes with the anti-BCAM antibody of the present invention for binding to human BCAM. Examples of assays that can be used include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), and sandwich competitive assay (see, for example, Stahli et al., (1983)). Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., (1986)). J. Immunol. 137:3614-9), solid-phase direct labeling assay, solid-phase direct labeling sandwich assay, Luminex (Jia et al., “A novel method of Multiplexed Competitive Antibody Binning for the characterization of monoclonal antibodies”) J. Immunological Methods(2004) 288, 91–98) and surface plasmon resonance (Song et al., “Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients”). J. Virol. (2010) 84, 6935-42).

[0140] Competitive binding can be determined using cells expressing human BCAM (e.g., 293 cells) by flow cytometry, for example, as shown in Example 4. Therefore, cells expressing human BCAM can be incubated with a saturated concentration (e.g., 20 µg / ml) of the unlabeled anti-BCAM antibody of the present invention sufficient to allow the anti-BCAM antibody of the present invention to bind to surface-expressed human BCAM for a period of time (e.g., 15-60 minutes). Control cells can be incubated under the same conditions without antibody or with a control antibody that does not bind human BCAM. At the end of the incubation period, the cells can be washed to remove any unbound antibody. A test antibody labeled with a detectable portion (e.g., a fluorescent portion) can be added to both the cells incubated with the anti-BCAM antibody of the present invention and the control cells for a period sufficient to allow the test antibody to bind to surface-expressed human BCAM for a period of time (e.g., 15-60 minutes). Subsequently, the cells can be washed, placed in a suitable culture medium (e.g., PBS containing 5% BSA), and analyzed by flow cytometry to quantify the number of labeled cells incubated with the anti-BCAM antibody of the present invention and the number of labeled control cells. If, compared to control cells, at least 10% (e.g., at least 50%) of the cells incubated with the unlabeled anti-BCAM antibody of the present invention are labeled with the test antibody, then the test antibody can be considered to compete with the anti-BCAM antibody of the present invention.

[0141] For example, another antibody may be provided that specifically binds to human BCAM and competes with antibodies containing the following amino acid sequences: HCDR1 containing the amino acid sequence of SEQ ID NO: 2, HCDR2 containing the amino acid sequence of SEQ ID NO: 3, HCDR3 containing the amino acid sequence of SEQ ID NO: 4; and a light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 5, LCDR2 containing the amino acid sequence of SEQ ID NO: 6, and LCDR3 containing the amino acid sequence of SEQ ID NO: 7 (e.g., MAb 477).

[0142] Alternatively, another antibody may be provided that specifically binds to human BCAM and competes with antibodies comprising: HCDR1 containing the amino acid sequence of SEQ ID NO: 8, HCDR2 containing the amino acid sequence of SEQ ID NO: 9, HCDR3 containing the amino acid sequence of SEQ ID NO: 10; and a light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 11, LCDR2 containing the amino acid sequence of SEQ ID NO: 12, and LCDR3 containing the amino acid sequence of SEQ ID NO: 13 (e.g., MAb 929).

[0143] Alternatively, another antibody may be provided that specifically binds to human BCAM and competes with antibodies comprising: HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, HCDR3 containing the amino acid sequence of SEQ ID NO: 20; and a light chain complementarity-determining region (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO: 23 (e.g., MAb 452).

[0144] In some cases, antibodies that specifically bind to human BCAM can be provided and compete with other antibodies (e.g., other antibodies) that contain the following: (A) The immunoglobulin heavy chain variable (VH) region containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14), and The immunoglobulin light chain variable (VL) region (i.e., the VH and VL regions of MAb 477) containing the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 15); and / or (B) The immunoglobulin heavy chain variable (VH) region containing the amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16), and The immunoglobulin light chain variable (VL) region (i.e., the VH and VL regions of MAb 929) contains the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

[0145] It can also provide specific binding to human BCAM and compete with other antibodies that bind to human BCAM, including those containing the following: The immunoglobulin heavy chain variable (VH) region containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO:24), and The immunoglobulin light chain variable (VL) region (i.e., the VH and VL regions of MAb 452) contains the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25).

[0146] Fc region The anti-BCAM antibody of the present invention may contain an Fc region. For various reasons, the anti-BCAM antibody described herein may have an Fc region. For example, the Fc region may provide one or more effector functions. Alternatively or additionally, an Fc region may be included to prolong the antibody's half-life in circulation.

[0147] The Fc region of the anti-BCAM antibody applicable to this invention includes the Fc region of an IgG subclass antibody (referred to herein as the "IgG Fc region"). The IgG Fc region may be human. The IgG Fc region may be humanized.

[0148] The IgG Fc region can be a subclass of IgG1. IgG1 can be human. The IgG Fc region can be a subclass of IgG3. IgG3 can be human.

[0149] Exemplary human IgG1 Fc contains the amino acid sequence ASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:76).

[0150] The Fc region can be modified to add one or more effector functions. For example, the Fc region can be modified to defucosylate.

[0151] The Fc region can be modified to reduce or eliminate one or more effector functions. In applications where anti-BCAM antibodies are primarily used to target the payload to BCAM-expressing cancer cells (e.g., when used in antibody-drug conjugates) or to activate the inherent cytolytic potential of T cells against BCAM-expressing cancer cells (e.g., when used in BiTE), effector functions may not be desired.

[0152] Mutations in the Fc region that lead to reduced or eliminated effector function include LALA mutations (commonly referred to as L234A / L235A, and optionally also K322A). Therefore, the Fc region may contain LALA mutations. Effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) are induced by the binding of antibodies to one or more Fc receptors (FcRs) such as FcRI, FcRII, and / or FcRIIIa. PG mutations (commonly referred to as P329G) can reduce or eliminate binding to FcRs. Therefore, in some embodiments, the Fc region contains PG mutations.

[0153] LALA and PG mutations can be combined, meaning the Fc region can contain both LALA and PG mutations. An example IgG1 Fc region containing both LALA and PG mutations is shown below, with LALA and PG mutations shown in bold: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 77).

[0154] Effector function The anti-BCAM antibodies described herein may include a functional Fc region, i.e., an Fc region that provides one or more effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC)).

[0155] Antibody-dependent cell-mediated cytotoxicity (ADCC) ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells, and the membrane surface antigens of the target cells are bound by specific antibodies.

[0156] When provided with a functional Fc region (e.g., as a full-length antibody), the anti-BCAM antibodies of the present invention (e.g., MAb232, 477, and 452) are capable of inducing measurable ADCC at nanomolar concentrations. ADCC can be evaluated as described in the examples provided herein, for example, by incubating BCAM-expressing cancer cells (e.g., OVSAHO or OAW28 cells) in the presence or absence of the antibody described herein, and measuring cell viability over a period of time in the presence or absence of the antibody. As demonstrated herein, antibodies capable of inducing ADCC can be used for antitumor therapy. Therefore, the anti-BCAM antibodies of the present invention are capable of inducing ADCC.

[0157] ADCC is mediated by the binding of IgG to Fc receptors (FcRs) on effector cells of the immune system, including natural killer (NK) cells, monocytes, macrophages, and eosinophils. The Fc receptors of IgG bind to FcRI, FcRII, and (primarily) FcRIIIa. For example, ADCC can be triggered by antibodies containing the Fc region of IgG1 or IgG3.

[0158] Therefore, the anti-BCAM antibodies described herein may contain a functional Fc region, such as an IgG Fc region. Such antibodies are capable of inducing ADCC. IgG may be a subclass of IgG1. IgG1 may be human (or humanized). Alternatively, IgG may be a subclass of IgG3. IgG3 may be human (or humanized).

[0159] The Fc region of IgG isotype antibody in each C H The N297 region of the 2-domain contains a conserved glycosylation site. N-linked oligosaccharides expressed at this site significantly influence the effector function of antibodies against the IgG isotype. For example, the presence of fucose residues can lead to severely reduced ADCC efficiency. Removal of the core fucose residue from the N-glycan of IgG-Fc results in a significant enhancement (>50-fold) of antibody-dependent cytotoxicity (ADCC) by improving the binding of IgG to FcR receptor IIIa (FcRIIIa) (Yamane-Ohnuki et al., 2004; Iida et al., 2009). Therefore, the Fc region of the anti-BCAM antibody described herein can be modified to be defucosylated.

[0160] The anti-BCAM antibody presented in this article can be defucosylated. Defucosylated antibodies can be used in functional groups lacking the encoding enzyme α-1,6-fucosyltransferase. FUT8 Antibodies produced in cell lines that express the gene or overexpress recombinant β-1,4-N-acetylglucosamine transferase III. Antibodies produced in these cell lines are rich in dimeric and non-fucosylated oligosaccharides.

[0161] Other antibody properties The anti-BCAM antibodies of the present invention (e.g., MAb 929, 477, and 452) exhibit excellent thermal stability, for example, when provided as full-length antibodies (e.g., in IgG form). Furthermore, they exhibit minimal multireactivity. They also exhibit minimal aggregation.

[0162] Thermostability can be checked using whole IgG antibodies. It is expected that the antibody will exhibit Fab stability >68°C during the thermostability test.

[0163] Multireactivity can be assessed by incubating the antibody with KLH, LPS, and / or human insulin, respectively. The antibodies described herein are expected to exhibit minimal multireactivity, such as no reaction with keyhole hemocyanin (KLH), lipopolysaccharide (LPS), and / or human insulin.

[0164] Furthermore, the anti-BCAM antibodies of the present invention (e.g., MAb 929, 477, and 452) do not contain sequence risk factors, including those that may be involved in aggregate formation. For example, two or more consecutive positively charged residues (e.g., RR or RKR) can contribute to the “stickiness” of the antibody, making them less ideal for therapeutic applications.

[0165] Aggregate formation can be assessed by incubating the antibody in a suitable storage buffer (e.g., PBS) at 4°C for 2–4 weeks (e.g., 4 weeks). Aggregation can be considered minimal when, for example, 10% or less of the antibody is present as high molecular weight aggregates at the end of the incubation period, as assessed by mass spectrometry.

[0166] Other sequence risk factors include amino acid residues that may undergo post-translational modifications (PTMs). PTMs can affect antibody affinity, stability, potency, and / or homogeneity. PTMs can include deamidation, isomerization, oxidation, N-linked glycosylation, free thiols, pyroglutamic acid, and C-terminal lysine.

[0167] Anti-BCAM antibodies may be free of sequence risk factors that interfere with the affinity, stability, potency, and / or uniformity of anti-BCAM antibodies, such as two or more consecutive positively charged residues or N-linked glycosylation.

[0168] The anti-BCAM antibody of the present invention satisfies at least the first two requirements listed in Table 3. The anti-BCAM antibody of the present invention satisfies at least the first three requirements listed in Table 3. Specifically, the anti-BCAM antibody of the present invention satisfies all the requirements listed in Table 3.

[0169] Table 3. Parameters Requirements Aggregation Form less than 10% aggregates after 4 weeks storage in PBS as assessed by mass photometry Thermal stability Have Fab stability of > 68 °C Polyreactivity Do not react with KLH, LPS, and human insulin Sequence risk factors Do not contain two or more consecutive positively charged residues or N-linked glycosylation that would interfere with affinity and / or stability Exemplary antibodies Provides a first-generation or humanized IgG antibody that specifically binds to human BCAM and contains: a) HCDR1 containing the amino acid sequence DYYVH (SEQ ID NO: 2), HCDR2 containing the amino acid sequence IINPFGGSATYAPKFQG (SEQ ID NO: 3), and HCDR3 containing the amino acid sequence GIYGHFDY (SEQ ID NO: 4); and b) LCDR1 containing the amino acid sequence KSSQSVLSASNNQNYLA (SEQ ID NO: 5), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 6), and LCDR3 containing the amino acid sequence QQYYSSPYT (SEQ ID NO: 7).

[0170] A second human or humanized IgG antibody is provided that specifically binds to human BCAM and comprises: a) HCDR1 containing the amino acid sequence SYYMH (SEQ ID NO: 18), HCDR2 containing the amino acid sequence WMNPNSGDSGYAQKFQG (SEQ ID NO: 19), and HCDR3 containing the amino acid sequence TYSSSWIFDY (SEQ ID NO: 20); and b) LCDR1 containing the amino acid sequence KSSQSVLYSSNNKNYLA (SEQ ID NO: 21), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 22), and LCDR3 containing the amino acid sequence QQYYSIPIT (SEQ ID NO: 23).

[0171] Provides a third-person or humanized IgG antibody that specifically binds to human BCAM and contains: a) HCDR1 containing the amino acid sequence SYAFS (SEQ ID NO: 8), HCDR2 containing the amino acid sequence GIIPFSGTTNYAQKFQG (SEQ ID NO: 9), and HCDR3 containing the amino acid sequence DPILSFWSGYYYYYGMDV (SEQ ID NO: 10); and b) LCDR1 containing the amino acid sequence RASQSISSWLA (SEQ ID NO: 11), LCDR2 containing the amino acid sequence DASNLET (SEQ ID NO: 12), and LCDR3 containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13).

[0172] A fourth human or humanized IgG antibody is provided, which specifically binds to human BCAM and contains: a) HCDR1 containing the amino acid sequence SYYMH (SEQ ID NO: 40), HCDR2 containing the amino acid sequence WINPNSGDTNYAQEFQG (SEQ ID NO: 41), and HCDR3 containing the amino acid sequence KRDGGGPFDY (SEQ ID NO: 42). b) LCDR1 containing the amino acid sequence KSSQSVLSRSNNKNYLA (SEQ ID NO: 43), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 44), and LCDR3 containing the amino acid sequence QQYYSTPFT (SEQ ID NO: 45).

[0173] Human or humanized IgG antibodies may meet at least the first three requirements listed in Table 2. In some cases, human or humanized IgG antibodies may meet at least the first three requirements and the last requirement listed in Table 2. Specifically, human or humanized IgG antibodies may meet all the requirements listed in Table 2.

[0174] Human or humanized IgG antibodies may meet at least the first two requirements listed in Table 3. In some cases, human or humanized IgG antibodies may meet at least the first three requirements listed in Table 3. Specifically, human or humanized IgG antibodies may meet all the requirements listed in Table 3.

[0175] If a human or humanized IgG antibody meets each of the requirements listed in Tables 2 and 3, it is considered particularly suitable for therapeutic use.

[0176] Human or humanized IgG antibodies may contain an Fc region (e.g., IgG1 or IgG3) capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing human BCAM. The Fc region may contain the amino acid sequence of SEQ ID NO: 76. In some cases, the Fc region does not contain the C-terminal K of the amino acid sequence of SEQ ID NO: 76.

[0177] Exemplary antibodies that can be used for the therapeutic applications disclosed herein are the human IgG antibodies listed in Table 1, specifically comprising: (i) HC containing the amino acid sequence of SEQ ID NO: 52 and LC containing the amino acid sequence of SEQ ID NO: 53; (ii) HC containing the amino acid sequence of SEQ ID NO: 50 and LC containing the amino acid sequence of SEQ ID NO: 51; and (iii) HC containing the amino acid sequence of SEQ ID NO: 64 and LC containing the amino acid sequence of SEQ ID NO: 65. Human IgG antibodies may contain one or more amino acid substitutions (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) that do not affect the binding properties of the parent antibody. For example, such antibodies may be modified to remove one or more sequence risk factors (e.g., two or more consecutive positively charged residues, such as RR or RKR). Depending on the desired therapeutic application, the Fc region of such antibodies may be modified to increase or decrease one or more effector functions. For example, the Fc region may be modified to be defucosylated. Alternatively or additionally, the Fc region can be modified to include LALA and PG mutations.

[0178] Bispecific antibodies The anti-BCAM antibody of the present invention can be included in a bispecific antibody. A bispecific antibody is a recombinant protein construct comprising a first antibody-binding component capable of specifically binding to a first antigen (e.g., a tumor-associated surface antigen, such as human BCAM) and a second antibody-binding component capable of specifically binding to a second antigen (e.g., a second tumor-associated surface antigen other than human BCAM). The first and second antibody-binding components are typically linked via a linker or adapter (e.g., a (flexible) peptide linker). In some cases, the bispecific antibody may comprise: a. An antibody component for binding human BCAM; b. Antibody components for binding to a second antigen other than human BCAM; c. Connecting an antibody component for binding human BCAM to a connecting component for binding other tumor-associated antigens; and optionally... d. Components used to extend half-life.

[0179] The anti-BCAM antibody of the present invention can be used in bispecific antibodies. Such antibodies include the aforementioned agonist and antagonist antibodies, as well as antigen-binding fragments derived therefrom, including suitable formatted single-chain antibodies.

[0180] The secondary antigen can be a tumor-associated surface antigen. Exemplary tumor-associated surface antigens that the antibody-binding component can bind to, besides human BCAM, include folate receptor-α (FR-α), PSA (prostate-specific antigen), HER2 (human epidermal growth factor receptor 2), MAGE (melanoma-associated antigen), CD33, CD30, CD22, CD79b, Nectin-4, TROP2, tissue factor (TF), CEA, immature laminin receptor, TAG-72, EpCAM, EphA3, mesothelin, SAP-1, gp100, TRP1, TRP2, MC1R, MUC1, EGFR, GD2, CD171, ErbB, PSMA, CDH6, Claudin6, transferrin receptor, PTK7, B7-H4, NaPI2b, MSLN, AXL, and ROR1. For example, overexpression of FR-α can be observed in more than 80% of ovarian cancers.

[0181] Various techniques for preparing bispecific antibodies are known to those skilled in the art. For example, the mortar and pestle technique involves engineering the CH3 domains of two different heavy chains to facilitate heterodimer formation. One CH3 domain is engineered to have a "mortar" mutation (typically replacing existing amino acid residues with amino acids containing large side chains, such as T366W), while the other CH3 domain is engineered to have a complementary "mortar" mutation (typically replacing existing amino acid residues with amino acids containing small side chains, such as T336S, L368A, Y407V). This spatially favors the pairing of the two different heavy chains, resulting in a bispecific antibody. The mortar and pestle technique allows for the production of full-length IgG-like bispecific antibodies with both native architecture and Fc-mediated effector function.

[0182] Another example is the CrossMab technique, which addresses the challenges associated with light chain mismatches in bispecific antibody preparation. The CH1 domain of a heavy chain is exchanged with the constant light (CL) chain domain of its corresponding light chain. This domain exchange ensures that each light chain pairs with its homologous heavy chain, thus preventing light chain mismatches. The resulting bispecific antibody maintains an IgG-like structure with correct heavy and light chain association. The CrossMab technique can be combined with the mortar and pestle technique or other Fc-engineered methods to further enhance bispecific antibody formation.

[0183] DuoBody technology involves generating two separate IgG1 antibodies using a recombinant mammalian expression system, each containing a single matching substitution (K409R and F405L) in the CH3 domain. After preparation and purification, the two antibodies are mixed under mild reducing conditions to perform Fab arm exchange, yielding a bispecific antibody. DuoBody technology allows for the efficient preparation of bispecific antibodies that are structurally and functionally very similar to the natural IgG1 molecule.

[0184] Bispecific antibodies can also be prepared in a direct manner by linking: (i) a first antibody that specifically binds to a first epitope on a first protein and (ii) a second antibody that specifically binds to a second epitope on either the first protein or a second (different) protein. The first and second antibodies can be linked via one or more linkers or adapters. At least one antibody may contain an Fc region. For example, one of the antibodies may be linked to an Fc region via a linker or adapter.

[0185] In some cases, the first and / or second antibodies can be reformatted to be included in a bispecific antibody. For example, a first full-length antibody binding to a first epitope can be reformatted as a single-chain antibody, such as scFv, and linked to the Fc region of a second full-length antibody binding a second epitope via a linker or adapter. For example, Coloma and Morrison ( Nat Biotechnol Articles 15, 159–163 (1997) describe the design and preparation of tetravalent bispecific antibodies in this manner. Alternatively, the first monodomain antibody and the second monodomain antibody may be linked to each other via a linker or adapter and attached to the Fc region to form a bispecific antibody. Many variations of combining the first and second antibodies in various ways to form bispecific antibodies are known to those skilled in the art.

[0186] Biparatopic antibodies Bispecific antibodies can specifically bind to a first epitope and a second (different) epitope. The first and second epitopes can be on different proteins. Alternatively, the first and second epitopes can be different epitopes on the same protein (e.g., on human BCAM). In some cases, the different epitopes are non-overlapping. Therefore, a bispecific antibody can be a biepisode anti-BCAM antibody. In some cases, a biepisode antibody may comprise: a. A first antibody component for binding to a first epitope on human BCAM; b. A second antibody component for binding to a second epitope on human BCAM; c. A connecting member for linking the first antibody component to the second antibody binding component; and optionally... d. Components used to extend half-life.

[0187] The first and second antibody components can be linked to the half-life extension molecule via peptide linkers. The half-life extension molecule may contain or consist of an Fc region. Alternatively, the half-life extension molecule may be a serum protein (e.g., human serum albumin).

[0188] The first antibody-binding component and the second antibody-binding component can be the anti-BCAM antibody of the present invention. Such antibodies include the above-described agonist antibodies and antagonist antibodies, as well as antigen-binding fragments derived therefrom, including suitable formatted single-chain antibodies.

[0189] For example, a biepisode anti-BCAM antibody may comprise a first antibody containing a VH region containing the amino acid sequence of SEQ ID NO: 16 and a VL region containing the amino acid sequence of SEQ ID NO: 17 (i.e., the VH and VL regions of MAb 929); and a second antibody containing a VH region containing the amino acid sequence of SEQ ID NO: 14 and a VL region containing the amino acid sequence of SEQ ID NO: 15 (i.e., the VH and VL regions of MAb 477).

[0190] In another example, the biepisode anti-BCAM antibody may comprise a first antibody containing a VH region containing the amino acid sequence of SEQ ID NO: 24 and a VL region containing the amino acid sequence of SEQ ID NO: 25 (i.e., the VH and VL regions of MAb 452); and a second antibody containing the amino acid sequence of SEQ ID NO: 14 and a VL region containing the amino acid sequence of SEQ ID NO: 15 (i.e., the VH and VL regions of MAb 477).

[0191] In another example, the biepisode anti-BCAM antibody may comprise a first antibody containing a VH region containing the amino acid sequence of SEQ ID NO: 24 and a VL region containing the amino acid sequence of SEQ ID NO: 25 (i.e., the VH and VL regions of MAb 452); and a second antibody containing a VH region containing the amino acid sequence of SEQ ID NO: 16 and a VL region containing the amino acid sequence of SEQ ID NO: 17 (i.e., the VH and VL regions of MAb 929).

[0192] Biepisode anti-BCAM antibodies may also contain an IgG Fc region, such as human IgG1 Fc containing the amino acid sequence SEQ ID NO: 76. In some cases, the IgG Fc region may be modified to contain one or more (e.g., two, three, or four) mutations (e.g., one or more deletions or substitutions). For example, the Fc region may contain one or more amino acid substitutions to promote heterodimer formation.

[0193] For example, a biepisode anti-BCAM antibody may comprise: (i) an HC of scFv and an LC of SEQ ID NO: 65 connected via a linker containing the amino acid sequence of SEQ ID NO: 115; (ii) an HC of scFv and an LC of SEQ ID NO: 51 connected via a linker containing the amino acid sequence of SEQ ID NO: 116; or (iii) an HC of scFv and an LC of SEQ ID NO: 51 connected via a linker containing the amino acid sequence of SEQ ID NO: 117.

[0194] Bi-specific T cell engagers (BiTEs) In some respects, a bispecific T-cell connector (BiTE) is provided. A BiTE is a recombinant protein construct comprising a first antibody-binding component capable of specifically binding to a selected tumor-associated surface antigen (e.g., human BCAM) and a second antibody-binding component targeting a surface-expressed subunit of the T-cell receptor complex (e.g., CD3). The first and second antibody-binding components are typically linked via a linker or adapter (e.g., a (flexible) peptide linker). BiTEs are designed to transiently connect T cells to target cells (e.g., cancer cells). This connection can activate the inherent cytolytic potential of T cells against target cells.

[0195] A bispecific T-cell adaptor (BiTE) may include: a) Antibody components for binding human basal cell adhesion molecule (BCAM); b) Antibody components for binding CD3; c) Connecting the antibody component for binding human BCAM to a connecting component for binding CD3; and optionally; d) Components used to extend half-life.

[0196] Antibody components for binding human BCAM may include anti-BCAM antibodies. For example, the anti-BCAM antibody of the present invention can be used in BiTE. Such antibodies include the aforementioned agonist and antagonist antibodies and antigen-binding fragments derived therefrom, including suitable formatted single-chain antibodies.

[0197] Antibody components for binding CD3 (such as anti-CD3 antibodies or their antigen-binding portions) are well known and include those described, for example, in US 2014 / 0302037, US 2014 / 0308285, WO 2008 / 119567, WO 2014 / 144722, WO2014 / 151910, WO 2015 / 048272, WO 2017 / 021354, and WO 2019 / 224717. Bonatetumab is an approved BiTE containing an anti-CD3 single-chain variable fragment (scFv) suitable for use as an antibody component for binding CD3.

[0198] BiTE may comprise two antibody-derived antigen-binding regions linked via (flexible) peptide linkers. A first antigen-binding region (e.g., a first antigen-binding fragment, such as a first scFv) is capable of specifically binding to selected tumor-associated surface antigens (e.g., human BCAM) on target cells (e.g., cancer cells expressing BCAM). A second antigen-binding region (e.g., a second antigen-binding fragment, such as a second scFv) is capable of specifically binding to CD3 on the surface of T cells.

[0199] BiTE may also contain a half-life extension molecule. The antigen-binding regions derived from the first and second antibodies can be linked to the half-life extension molecule via peptide linkers. The half-life extension molecule may contain or consist of an Fc region. Alternatively, the half-life extension molecule may be a serum protein (e.g., human serum albumin).

[0200] The first antigen-binding region targeting human BCAM can be derived from any anti-BCAM antibody described herein. Such antibodies include the aforementioned agonist and antagonist antibodies, as well as antigen-binding fragments derived therefrom, including suitable formatted single-domain and single-chain antibodies.

[0201] The exemplary first antigen-binding regions derived from MAb 477, 929, and 452 respectively contain one of the following anti-BCAM scFv: (A) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 78); (B) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 79); and (C) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 82).

[0202] Exemplary second antigen binding regions capable of specifically binding CD3 on the surface of T cells comprise one of the following anti-CD3 scFvs: (A) DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGE GTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIK (SEQ ID NO: 83); and (B) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE GGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 111).

[0203] An example of a half-life-extending molecule is the following IgG Fc region containing LALA and PG mutations: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 77).

[0204] The peptide linkers applicable to BiTE described herein contain or consist of the amino acid sequences GGGG (SEQ ID NO: 26) and GGGGS (SEQ ID NO: 27).

[0205] The signal peptide applicable to the BiTE described herein comprises or consists of the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 84).

[0206] Chimeric antigen receptors In some respects, chimeric antigen receptors (CARs) are provided. CARs typically include components for specifically binding to tumor-associated surface antigens (e.g., human BCAM) on target cells (e.g., cancer cells expressing BCAM), a transmembrane domain, and a cytoplasmic domain comprising a signal transduction domain and optionally at least one co-stimulatory signal transduction domain.

[0207] The binding component can be an antibody (e.g., an antigen-binding fragment, such as scFv) capable of specifically binding to a tumor-associated surface antigen (e.g., human BCAM) on target cells (e.g., cancer cells expressing BCAM). The binding component can be derived from any anti-BCAM antibody described herein. Such antibodies include the aforementioned agonist and antagonist antibodies and antigen-binding fragments derived therefrom, including suitable formatted single-domain and single-chain antibodies.

[0208] Exemplary bonding components derived from MAb 477, 929, and 452 respectively include the following anti-BCAM scFv: (A) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGS GGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 78); (B) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 79); and (C) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 82).

[0209] The transmembrane domain may be derived from the α, β, or ζ chain of the T cell receptor (TCR), NKG2D, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154 (e.g., containing at least its transmembrane region). In some cases, the transmembrane domain may be synthetic and primarily contains hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan, and / or valine may be present at each end of a synthetic transmembrane domain. In other cases, the transmembrane domain may be derived from CD8 or CD28.

[0210] Upon binding to a tumor-associated surface antigen, the signal transduction domain contained in the cytoplasmic domain of the CAR activates one or more effector functions of the immune effector cells (e.g., T cells) expressing the CAR. T cell effector functions may include, for example, cytolytic activity or helper activity, including cytokine secretion.

[0211] The signal transduction domain may originate from DAP10, DAP12, Fc ε receptor I γ chain (FCER1G), FcR β, NKG2D, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, or CD79B (e.g., containing at least their signal transduction domains). For example, the cytoplasmic domain may contain the CD3ζ signal transduction domain.

[0212] The cytoplasmic domain may also contain one or more co-stimulatory signal transduction domains. One or more co-stimulatory signal transduction domains may originate from CD28, 41BB, IL2Rb, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, or HVEM (e.g., containing at least their signal transduction domains). For example, a co-stimulatory signal transduction domain may originate from CD28.

[0213] Cells capable of expressing CARs as described herein, including immune effector cells, are also provided. As used herein, the term "immune effector cell" refers to a cell differentiated into a form capable of regulating or achieving a specific immune response. Such cells may include mature lymphocytes suitable for therapy, including but not limited to cytotoxic T cells, helper T cells, natural killer cells (NK cells), and tumor-infiltrating lymphocytes (TILs), and may also include dendritic cells or macrophages. For example, an immune effector cell may be a T cell. Alternatively, an immune effector cell may be an NK cell.

[0214] Drug conjugates In some respects, antibodies are conjugated to drugs. Typically, drug conjugates are provided that comprise a binding component (e.g., a recombinant protein) capable of specifically binding to a selected tumor-associated surface antigen (e.g., human BCAM), which is then linked via a linker to a component for killing tumor cells (e.g., a cytotoxic drug).

[0215] Antibody-drug conjugates (ADCs) are provided, which may comprise an antibody (e.g., a full-length antibody and / or an antigen-binding fragment, such as scFv) linked via a linker to a drug (also referred to as a "payload"). Typically, the drug is a cytotoxic agent, for example, used to treat cancer. The anti-BCAM antibody of the present invention can be used in an ADC. For example, an ADC may comprise a bispecific antibody as described herein. The bispecific antibody may be a biepitaxy anti-BCAM antibody as described herein.

[0216] In the development of ADC-based anti-tumor therapies, anticancer drugs (e.g., cytotoxins) are conjugated to antibodies that specifically target and bind to tumor markers expressed on the surface, such as BCAM on cancer cells. This complex is internalized via receptor-mediated endocytosis, and the cytotoxic drug is released and kills the cancer cells. Release may be due to proteolytic degradation of the antibody, such as in lysosomes, or due to cleavage of the linker.

[0217] By combining the targeting capabilities of antibodies with the cancer-killing abilities of cytotoxic drugs, antibody-drug conjugates (ADCs) allow for the differentiation between healthy and diseased tissues. Compared to administering cytotoxic drugs alone, ADCs target cancer cells, resulting in less impact on healthy cells, thus reducing side effects and providing a wider therapeutic window.

[0218] Linker Cleavable and non-cleavable linkers have been shown to be safe in both preclinical and clinical trials. For example, brentuximab vedotin (ADCETRIS) contains an enzyme-sensitive cleavable linker that delivers the potent and highly toxic anti-microtubule agent monomethylaurestatin E (MMAE) (a synthetic antitumor agent) to CD30-positive tumor cells. Due to its high toxicity, MMAE, which inhibits cell division by blocking microtubule polymerization, cannot be used alone as a chemotherapy drug. ADCs in which MMAE is linked to an anti-CD30 monoclonal antibody have been shown to be stable in the extracellular fluid and therefore safe for treatment. Detrastuzumab (ENHERTU) contains delutecan, a topoisomerase I inhibitor linked to trastuzumab via a cathepsin-cleavable GGFG (SEQ ID NO: 85) tetrapeptide-aminomethoxy linker. Trastuzumab emtansine (KADCYLA) is another approved ADC, which is a combination of the microtubule formation inhibitor metansine (DM-1) (a derivative of maytansine) and the antibody trastuzumab via an uncuttable linker conjugate.

[0219] Linkers can contain disulfides, hydrazones, thioethers, or peptides. Peptide-based linkers can become cleavable by incorporating sites that are cleaved by enzymes present in endosomes or lysosomes (e.g., cathepsins). A well-known cleavable linker is valine-citrulline-p-aminobenzylcarbamate. Linking cytotoxic agents to antibodies can prevent extracellular cytotoxic activity, thereby reducing off-target effects. Cytotoxic payloads delivered via cleavable linkers can escape from targeted cells and destroy neighboring cancer cells in a process known as “bystander killing.”

[0220] Therefore, drugs can be conjugated to antibodies via cleavable linkers. Cleavable linkers can be enzyme-cleavable linkers. Enzyme-cleavable linkers can be peptide-based linkers. Enzyme-cleavable linkers can contain protease cleavage sites. Proteases can be lysosomal enzymes (e.g., cathepsins). For example, peptide-based cleavable linkers can contain the amino acid sequence GGFG (SEQ ID NO: 85). Specifically, a cleavable GGFG (SEQ ID NO: 85) tetrapeptide-aminomethoxy linker can be used in the ADCs described herein.

[0221] Alternatively, the drug can be conjugated to the antibody via an uncuttable linker.

[0222] Drug Typically, the drugs in ADCs are anticancer drugs that reduce or weaken the activity of cancer cells that have internalized them.

[0223] Anticancer drugs can be cytotoxic agents. Cytotoxic agents can be tubulin inhibitors or DNA damaging agents. Tubulin inhibitors can be aurestatin (e.g., MMAE), maytansine alkaloids (e.g., maytansine or DM-1), or tubulolysin. DNA damaging agents can be agents that induce DNA double-strand breaks, DNA alkylating agents, topoisomerase inhibitors, or DNA cross-linking agents. Agents that induce DNA double-strand breaks can be calichimycin. DNA alkylating agents can be docamycin. Topoisomerase inhibitors can be eczemab (e.g., delutecan). DNA cross-linking agents can be pyrrolobenzodiazepines.

[0224] Anticancer drugs can be immunomodulators. Immunomodulators can be TLR agonists or STING agonists.

[0225] Anticancer drugs can be toxins. Toxins can be selected from ricin, abrin, alpha toxin, saponins, ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, white tree toxin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0226] Anticancer drugs can be radioactive nuclides. Radioactive nuclides can be those that emit alpha particles. Exemplary alpha-emitting radioactive nuclides include... 211 At、 212 Bi、 212 Pb, 213 Bi、 223 Ra、 225 Ac and 227 The radionuclide can be linked to the antibody via a chelate-containing linker (e.g., a derivative of DTPA or DOTA).

[0227] An ADC may contain one drug. Alternatively, an ADC may contain more than one drug, such as two different drugs. Two or more different drugs may have different modes of action. Alternatively, two or more different drugs may have the same mode of action. ADCs containing two or more drugs may be particularly effective in cancer therapy (see, for example, Yamazaki et al., 2021). Nat. Comms. 12(3528)).

[0228] Drug-antibody ratio (DAR) In ADCs, the drug-antibody ratio (DAR) typically refers to the average number of drug molecules conjugated to the antibody. The optimal DAR for a given ADC can vary, for example, depending on the type of antibody, drug, and / or conjugation site used.

[0229] Typically, DAR ranges from about 1 to about 20. DAR can range from about 1 to about 16. DAR can range from 1 to 10 (e.g., about 4-8). DAR can be about 1, about 2, about 4, about 6, about 8, or about 10. As demonstrated in this paper, a DAR of 4 is suitable for achieving the desired therapeutic effect.

[0230] Nucleic acids, expression vectors, and host cells Nucleic acids and expression vectors encoding the anti-BCAM antibody, BiTE, or CAR of the present invention are provided, as well as host cells comprising the nucleic acid or expression vector encoding the anti-BCAM antibody, BiTE, or CAR described herein. Exemplary nucleic acids encoding the HC and LC regions of the anti-BCAM antibodies of Table 1 are provided in Table 4.

[0231] Table 4. Anti-BCAM antibody nucleic acid sequences for HC and LC regions Antibody Heavy chain constant SEQ ID NO Light chain constant SEQ ID NO 228 86 87 232 88 89 452 90 91 477 92 93 606 94 95 929 96 97 937 98 99 In some cases, a single nucleic acid encoding the anti-BCAM antibody, BiTE, or CAR of the present invention is provided. An expression vector comprising this nucleic acid may also be provided.

[0232] In other cases, a first nucleic acid and a second nucleic acid are provided, wherein: a) The first nucleic acid contains a variable immunoglobulin heavy chain (V) encoding the anti-BCAM antibody of the present invention. H The nucleotide sequence of the region, and b) The second nucleic acid contains a variable (V) light chain of an immunoglobulin encoding the same antibody. L The nucleotide sequence of the region.

[0233] A first expression vector and a second expression vector, each containing a first nucleic acid and a second nucleic acid, are also provided. Alternatively, the first and second nucleic acids may be contained in a single expression vector.

[0234] It also provides host cells containing nucleic acids or expression vectors.

[0235] Methods for preparing the provided anti-BCAM antibody, BiTE, or CAR are also provided. The method includes providing a host cell containing one or more nucleic acids (e.g., expression vectors) encoding the anti-BCAM antibody, BiTE, or CAR of the present invention, and incubating the host cell under conditions suitable for expressing the antibody, BiTE, or CAR encoded by the one or more nucleic acids. The one or more nucleic acids (or expression vectors) may comprise a first nucleic acid (or expression vector) encoding the amino acid sequence of the light chain of the antibody described herein and a second nucleic acid (or expression vector) encoding the amino acid sequence of the heavy chain of the antibody.

[0236] For expression in suitable host cells, the anti-BCAM antibody, BiTE, or CAR of the present invention may have a suitable signal peptide. This signal peptide may comprise or consist of the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 84) or MGWSCIILFLVATATGVHS (SEQ ID NO: 100).

[0237] Pharmaceutical Composition This article provides a pharmaceutical composition comprising an antibody that specifically binds to human basal cell adhesion molecule (BCAM), the antibody comprising a variable region and a constant region, wherein the variable region comprises a framework region and a complementary determining element for binding human BCAM, the antibody being mixed with a pharmaceutically acceptable carrier.

[0238] For example, anti-BCAM antibodies, anti-BCAM drug conjugates (e.g., anti-BCAM ADC), anti-BCAM BiTE, and (immune effector) cells capable of expressing the anti-BCAM CAR described herein can be formulated into pharmaceutical compositions.

[0239] A pharmaceutical composition may contain one or more of a pharmaceutically acceptable carrier, diluent, or excipient.

[0240] Pharmaceutically acceptable carriers can be conventional (e.g., as described in Remington, The Science and Practice of Pharmacy, 22nd ed., Loyd V., Pharmaceutical Press, 2012). Generally, the nature of the carrier depends on the mode of administration. For example, parenteral preparations typically contain an injectable fluid as a medium, including pharmaceutically and physiologically acceptable fluids such as water for injection, physiological saline, balanced salt solutions, etc. The pharmaceutical composition may additionally contain small amounts of non-toxic excipients (e.g., one or more buffers) to achieve stability.

[0241] The carrier may be sterile and / or suspended or otherwise contained in a unit dosage form comprising one or more measured doses of a composition suitable for administering an effective amount of the antibody described herein to a subject. Medicinal products for treatment may also be included in such embodiments. The unit dosage form may be in a sealed vial containing sterile contents or in a syringe for injection into a subject, lyophilized for subsequent dissolution and administration. The pharmaceutical composition may be provided in a solid or controlled-release dosage form.

[0242] Pharmaceutical compositions typically contain an “effective” or “therapeuticly effective” amount (as used interchangeably herein) of the antibody described herein. The dosage and dosing regimen for achieving the desired therapeutic outcome can depend on the route of administration. They can vary based on factors including, but not limited to, the individual’s disease state, age, sex, and weight, and the antibody’s ability to elicit the desired response in the individual. An effective amount is also the amount in which the beneficial therapeutic effect exceeds any toxic or adverse effects of the antibody described herein.

[0243] therapy This article provides a method for treating cancer in human subjects, comprising administering to the human subject an effective amount of an antibody that specifically binds to human basal cell adhesion molecule (BCAM), the antibody comprising a variable region and a constant region, wherein the variable region comprises a framework region and a complementary determining component for binding human BCAM, the antibody being mixed with a pharmaceutically acceptable carrier.

[0244] For example, the anti-BCAM antibodies, anti-BCAM ADCs, and anti-BCAM BiTEs described herein, as well as pharmaceutical compositions comprising them, can be used in therapies, particularly in methods for treating cancers (e.g., ovarian or endometrial cancer) in their subjects. Similarly, anti-BCAM CARs and immune effector cells expressing such CARs can be used in methods for treating cancers in subjects in need. Specifically, the use of anti-BCAM antibodies, anti-BCAM ADCs, anti-BCAM BiTEs, anti-BCAM CARs, and (immune effector) cells capable of expressing the anti-BCAM CARs described herein in the manufacture of medicaments for treating cancer is envisioned.

[0245] A method for treating cancer in a subject in need is provided, wherein the method includes administering to the subject a therapeutically effective amount of a component for specifically binding human BCAM and modulating the binding of human BCAM to laminin α-5. Furthermore, a method for treating cancer in a subject in need is provided, wherein the method includes administering to the subject a therapeutically effective amount of a component for specifically binding human BCAM and inhibiting the binding of human BCAM to laminin α-5. Additionally, a method for treating cancer in a subject in need is provided, wherein the method includes administering to the subject a therapeutically effective amount of a component for specifically binding human BCAM and increasing the binding of human BCAM to laminin α-5.

[0246] Furthermore, a method for treating cancer in a subject in need is provided, wherein the method includes administering to the subject a therapeutically effective amount of a component for specifically binding human BCAM and selectively inducing cell death in BCAM-expressing cancer cells. Cell death may be induced, for example, through one or more effector functions of the binding component, through a cytotoxic component (e.g., one or more cytotoxic drugs) attached to the binding component, or by inducing immune effector cells (e.g., T cells or NK cells) to kill BCAM-expressing cancer cells.

[0247] A method for treating cancer in a subject in need is also provided, wherein the method includes administering to the subject a therapeutically effective amount of a bispecific T-cell connective (BiTE), the bispecific T-cell connective comprising a first binding component capable of specifically binding to human BCAM and a second binding component targeting a surface-expressed subunit (e.g., CD3) of a T-cell receptor complex, wherein the first and second binding components are connected via a peptide linker. Furthermore, a method for treating cancer in a subject in need is provided, wherein the method includes administering to the subject a therapeutically effective amount of a drug conjugate (e.g., an ADC), the drug conjugate comprising a binding component capable of specifically binding to human BCAM and a component for killing tumor cells (e.g., a cytotoxic drug), wherein the binding component and the killing component are connected via a linker (e.g., a cleavable linker).

[0248] Therapeutic effective doses can be administered in dosing regimens that may contain multiple unit doses. Therapeutic effective doses (and / or appropriate unit doses within an effective dosing regimen) can vary, for example, depending on the route of administration and combination with other agents. Furthermore, the specific therapeutic effective dose (and / or unit dose) for any particular subject can depend on a variety of factors, including the condition being treated and its severity; the activity of the specific agent used; the specific composition used; the subject's age, weight, general health condition, sex, and diet; the timing, route of administration, and / or rate of excretion or metabolism of the specific therapeutic molecule used; the duration of treatment; and similar factors well-known in the medical field.

[0249] The clinical efficacy of the therapeutic treatments described in this article can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by the sum of the percentage of patients achieving a complete response (CR), the number of patients achieving a partial response (PR), and the number of patients with stable disease (SD) at a time point at least 6 months after the end of treatment. The CBR for a specific treatment regimen can be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Efficacy can also be assessed by improvements in key cancer endpoints, overall survival (OS), disease-free survival (DFS), objective response rate (ORR), complete response rate (CRR), or progression-free survival (PFS). See also Dept. of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics: Guidance for Industry (2018); E.A. Eisenhauer et al., New Response Evaluation Criteria in Solid Tumors: Revised RECIST Guideline (1.1 version), 45 Eur. J. Cancer 228 (2009). The degree of improvement in these clinically meaningful endpoints will vary depending on the cancer being treated and other considerations of the attending physician. OS or PFS can be an expected relative improvement of at least 20% or at least 25% compared to that achieved with standard of care and an absolute increase in PFS and / or OS for at least 2.5 months (e.g., 6 months). See Kumar et al., An Appraisal of Clinically Meaningful Outcomes Guidelines for Oncology Clinical Trials , JAMA Oncol. 2016 Sep 1; 2(9): 1238–1240; Ellis et al., American Society of Clinical Oncology perspective: Raising the bar for clinical trials by defining clinically meaningful outcomes , J Clin Oncol. April 20, 2014; 32(12):1277-80.

[0250] Cancer can be categorized into several groups, including ovarian cancer, endometrial cancer, colorectal cancer, breast cancer, prostate cancer, lung cancer, liver cancer, thyroid cancer, head and neck cancer, stomach cancer, pancreatic cancer, urothelial carcinoma, cervical cancer, skin cancer, and bladder cancer. In some cases, the cancer may be a sarcoma, carcinoma, or basal cell carcinoma. Specifically, cancer can be ovarian cancer or endometrial cancer. Ovarian cancer can be high-grade ovarian cancer. Ovarian cancer can be serous ovarian cancer.

[0251] The treatment methods described herein may include administering a therapeutically effective amount of an anti-BCAM antibody, an anti-BCAM bispecific T-cell connective (BiTE), or an anti-BCAM antibody-drug conjugate (ADC) (e.g., formulated as a pharmaceutical composition as described herein) to a subject requiring such treatment. The anti-BCAM antibody, BiTE, or ADC may contain an IgG Fc region. The IgG Fc region may be human. The IgG Fc region may be humanized. The IgG Fc region may be IgG1, such as human IgG1.

[0252] The subject may be treated with one or more additional therapies. One or more additional therapies may include radiation therapy. One or more additional therapies may include chemotherapy. One or more additional therapies may include surgery. Alternatively, the subject may be untreated. In some cases, the subject may be treated first with one or more additional therapies. Alternatively, the subject may be treated first with anti-BCAM antibodies, anti-BCAM BiTE, or anti-BCAM ADCs (e.g., to shrink the tumor before surgery).

[0253] Anti-BCAM antibodies, anti-BCAM BiTE, or anti-BCAM ADCs may be administered sequentially to the subject in conjunction with one or more other therapeutic agents (e.g., immune checkpoint inhibitors). The other therapeutic agents may be administered to the subject before, during, or after administration of the anti-BCAM antibody, anti-BCAM BiTE, or anti-BCAM ADC.

[0254] Example The following examples are for illustrative purposes only and are not intended to limit the scope of the appended claims.

[0255] Example 1. Identification and characterization of anti-BCAM antibodies This example demonstrates the identification and characterization of anti-BCAM antibodies.

[0256] Screening of fully human scFv phage display libraries (SuperHuman 2.0 antibody library, Distributed Bio / Charles River Labs) to identify antibodies binding to human BCAM was similar to that used by Schrofelbauer et al. (2022). PNAS The process is described in 120(1)1-11 and in published international patent application WO2023 / 114543. Thirty-seven fully human antibodies were identified as specific for human BCAM. The identified antibodies were characterized by determining their affinity for human BCAM expressed on cells, their cross-reactivity with cynomolgus monkey and mouse BCAM, their binding to BCAM knockout cells, and their cross-reactivity with homologous proteins, namely receptor for advanced glycation end products (RAGE), melanoma cell adhesion molecule (MCAM), and activated leukocyte adhesion molecule (ALCAM)). The results of these studies are summarized in Table 6. In Table 6, “Nd” indicates “not determined”.

[0257] Table 6. Characterization of anti-BCAM human antibodies.

[0258] The binding affinity of the antibody to human BCAM expressed on the cells was measured using flow cytometry. Expi293 cells transfected with human BCAM were washed with phosphate-buffered saline (PBS) and harvested using a CellStripper (Thermo Fisher). Antibody staining was performed in PBS / 5% bovine serum albumin (BSA), with the amount of primary antibody as indicated in the manufacturer's datasheet. Additional staining was performed with the appropriate fluorescently labeled secondary antibody. All washing steps were performed in PBS / 5% BSA.

[0259] For some antibodies, the binding affinity to recombinant human BCAM was also determined using ELISA. 20 µg / ml of recombinant BCAM (Sino) was coated onto a 96-well high-absorption plate and incubated overnight at 4°C. The plate was washed twice with PBS (Thermo Fisher) and blocked with PBS / 10% BSA for 1 hour at room temperature. Primary antibody in PBS / 10% BSA was added at serial dilutions from 10 µg / ml to 0.01 µg / ml and incubated for 1 hour at room temperature. The plate was washed three times with PBS / 0.1% Tween, and then anti-human HRP secondary antibody (Cell Technologies) diluted 1:1000 in PBS / 10% BSA was added and incubated for 30 minutes at room temperature. The sample was washed three times with PBS / 0.1% Tween, and TMB substrate (Pierce) was added to the wells for 15 minutes at room temperature. 2M sulfuric acid was added directly to the TMB substrate to terminate the reaction. The absorbance at 450 nm was recorded on a SpectraMax M5E (Molecular Devices).

[0260] To assess cross-reactivity, Expi293 cells were transfected with human BCAM, cynomolgus monkey BCAM, mouse BCAM, RAGE, MCAM, and ALCAM cDNA constructs, respectively. Overexpression of the corresponding proteins was validated using commercially available anti-BCAM, anti-RAGE, anti-MCAM, and anti-ALCAM antibodies, respectively. Flow cytometry was used to measure antibody binding to each protein. Antibodies were classified as cross-reactive with cynomolgus monkey and mouse BCAM, as well as the BCAM homologs RAGE, MCAM, and ALCAM, when >5% binding was observed.

[0261] To evaluate nonspecific cell binding, flow cytometry was performed after incubating the test antibody with both the Kuramochi Cas9 sgControl cell line (positive control) and the Kuramochi Cas9-sghBCAM knockout cell line (negative control). Antibodies were characterized as binding to BCAM knockout cell lines when >2.5% binding was observed.

[0262] No further studies were conducted on antibodies that showed cross-reactivity with one or more of RAGE, MCAM, and ALCAM.

[0263] Example 2. Epitope mapping of anti-BCAM antibody This example demonstrates additional biochemical and biophysical properties of a subset of 15 of the 37 anti-BCAM antibodies identified in Example 1.

[0264] Human BCAM / MCAM chimeras were used to plot the binding epitopes of anti-BCAM antibodies. A schematic diagram of the chimeras used in these experiments is shown below. Figure 1 Cells were transfected with cDNA of the corresponding chimeric, BCAM, or MCAM variants. Antibody binding was measured by flow cytometry after staining with test antibodies and anti-human APC secondary antibody. The results of these experiments are summarized in Table 7.

[0265] Table 7. Characterization of the combination of BCAM and MCAM.

[0266] Antibody Combined with 51 BCAM, B2, B3, B4, M4 (weak) 16 BCAM, M1, B3, B4, M4 (weak) 48 BCAM, B2, B3, B4, M4 (weak) 154 BCAM, B2, B3, B4 600 BCAM, B2, B3, B4, M4 (weak) 609 BCAM, B2, B3, B4, M4 (weak) 236 BCAM, B2, B3, B4 929 BCAM, M1, B2, B3, B4, M4 (weak) 604 BCAM, B2, B3, B4 606 BCAM, B2, B3, B4 941 BCAM, B2, B3, B4 330 BCAM, M1, B2, B3, B4, M4 (weak) 174 BCAM, B2, B3, B4, M4 (weak) 424 BCAM, B2, B3, B4, M4 (weak) 220 M1 452 BCAM, M1, B3, B4 477 BCAM, M1, B2, B3, B4 228 BCAM, B2, B3, B4 232 BCAM, M1, B2, B3, B4 937 BCAM, M1, B2, B3, B4 An illustrative histogram of flow cytometry assays using antibodies 929, 477, and 452 is shown in [the figure]. Figure 2A , Figure 2B and Figure 2C These histograms show that antibodies 929 and 477 require the BCAM V1-V2 domains for binding. In contrast, 452 requires both the BCAM V2 and C1 domains for binding. These domains overlap with the laminin α-5 binding domain. Antibodies cross-reactive to mouse BCAM were excluded from further investigation.

[0267] Example 3. Biophysical characterization of the selected anti-BCAM antibody Further biophysical characterization was performed on antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937.

[0268] thermal stability The thermostability of antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 was examined using whole IgG antibodies. All antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 showed Fab stability >68°C, indicating that the antibodies are thermostable.

[0269] Aggregation Mass spectrophotometry was used to determine whether antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 aggregated after purification. The results of this analysis are summarized in Table 8.

[0270] Table 8. Fractions of the purified 150 kDa antibody.

[0271] Antibody A series of approximately 150 kDa 228 97% 232 83% 236 96% 330 88% 452 96% 477 92% 606 82% 929 96% 937 89% All antibodies showed a molecular weight distribution of approximately 150 kDa (natural antibody weight). For most of the antibodies tested, only very small amounts (e.g., 10% or less) of high molecular weight aggregates were detected.

[0272] Of the antibodies tested, 228, 236, 452, 477, and 929 had the smallest amount (10% or less) of high molecular weight aggregates.

[0273] Example 4. Functional characterization of the selected anti-BCAM antibody Antibodies 228, 232, 236, 330, 452, 477, 606, 929 and 937 were further characterized functionally.

[0274] Multireactivity Using ELISA, the reactivity of antibodies 228, 232, 236, 452, 477, 606, 929, and 937 with keyhole hemocyanin (KLH), lipopolysaccharide (LPS), and human insulin was also tested. Lenzrucumab was used as a positive control because it reacts with insulin. None of the antibodies tested reacted with KLH, LPS, or human insulin.

[0275] Antibody-dependent cytotoxicity The ability of antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 to induce antibody-dependent cytotoxicity (ADCC) was tested. Each of the nine antibodies was added at various concentrations to either the human high-grade serous ovarian cancer cell line OVSAHO or the human ovarian serous cystadenocarcinoma cell line OAW28, both co-cultured with the Jurkat-NFAT-Luc reporter cell line. Both OVSAHO and OAW28 expressed high levels of human BCAM. Luciferase signal was used as the readout value for ADCC activity. The fold changes in ADCC activity detected in the presence of various concentrations of the test antibodies are shown below. Figure 3A and Figure 3B Antibodies 232, 477, and 452 were able to induce measurable ADCC in both OVSAHO and OAW28 cells at nanomolar concentrations (e.g., down to 0.3 nM). Antibodies 937 and 606 also showed some activity, but only at the higher end of the tested nanomolar concentrations, such as approximately 40 nM.

[0276] Antibodies 452, 477, 606, 929, and 937 were further tested using the SKOV3 cell line expressing recombinant BCAM (SKOV3-BCAM), co-cultured with the same Jurkat-NFAT-Luc reporter cell line at various antibody concentrations. SKOV3 is a human ovarian cancer cell line with an epithelial-like morphology that typically does not express high levels of BCAM. As a positive control, corresponding experiments were performed with various concentrations of the anti-Her2 antibody trastuzumab. The luciferase signal (expressed as relative light units) was used as the readout value for natural killer (NK) cell activation. Results for each test antibody and trastuzumab are shown in [Table / Insert table here]. Figure 3C In the study, antibodies 452 and 477 induced ADCC at concentrations of 1 nM or lower with levels similar to trastuzumab. Antibodies 606, 929, and 937 also induced ADCC at antibody concentrations exceeding 1 nM.

[0277] Effects on BCAM:Laminin Interactions The effects of antibodies 228, 232, 236, 330, 452, 477, 606, 929, and 937 on the interaction between human BCAM and its native ligand laminin α-5 were tested using ELISA. ELISA plates were coated with recombinant laminin α-5. BCAM binding was tested in the presence or absence of each test antibody. Various antibody concentrations were tested. The results of these experiments are summarized in […]. Figure 4 middle.

[0278] Antibodies 228 and 606 did not affect the binding of laminin α-5 to BCAM. Antibodies 452 and 236 increased the binding of BCAM to laminin α-5 (i.e., acted as agonists), while the presence of antibodies 330, 937, 929, 477, and 232 decreased the binding (i.e., acted as antagonists). These results indicate that antibodies 228 and 606, 452 and 236, and 232, 330, 477, 929, and 937 bind to different epitope regions on human BCAM. These results also suggest that the binding of anti-BCAM antibodies to each of these epitope regions affects the ability of human BCAM to bind laminin α-5 in different ways.

[0279] Competition Measurement Method Antibodies 452, 477, and 929 competed for binding with each other and with each of antibodies 228, 232, 236, 606, and 937.

[0280] Label the antibody with Alexa Fluor 488 according to the manufacturer's instructions (Thermo Fisher). For staining, use 3 × 10⁻⁶ ppm. 5Cells were aliquoted into individual wells. For blocking, unlabeled antibody was added to the sample at a saturation level (20 mg / ml) and incubated on ice for 30 minutes. Control cells were incubated either without antibody or with an unrelated control antibody. After washing, Alexa Fluor 488-labeled antibody was added to both the blocking and control samples for 30 minutes. After washing, cells were resuspended in PBS / 5% BSA for flow cytometry analysis. Fluorescence activated cell sorting (FACS) analysis was performed on a BD Fortessa as described by Schröfelbauer et al. (PNAS, January 2023). The results of these experiments are summarized in Figure 5A , Figure 5B and Figure 5C middle.

[0281] from Figure 5A As can be seen, no competition was detected between antibody 452 and antibodies 477 and 929, indicating that their epitopes do not overlap. This observation is consistent with the finding that antibody 452 binds to different epitopes located in the V2 and C1 domains of human BCAM. It was also found that antibody 452 binds to epitopes different from those bound by antibodies 228, 232, 236, 606, and 937. Notably, antibody 452 does not compete with antibody 236 for binding to human BCAM, even though both antibodies increase the binding of laminin α-5 to BCAM.

[0282] like Figure 5B As illustrated, antibodies 232 and 477 compete with antibody 929 for binding to human BCAM, consistent with the finding that all three antibodies are able to reduce the binding of laminin α-5 to BCAM.

[0283] Similarly, antibody 477 was found to competitively bind to BCAM with antibody 232 (see [link to study]). Figure 5C Interestingly, no competition was observed between antibodies 477 and 929 in this experiment, indicating that these two antibodies can bind to adjacent but non-overlapping epitopes in the V1-V2 domains of human BCAM.

[0284] Example 5. Binding characteristics of anti-BCAM antibody Further analysis was conducted on the binding characteristics of antibodies 452, 477, and 929.

[0285] Characterization of the role of point mutations To further map the amino acids involved in the binding of these antibodies to human BCAM, each antibody was incubated with 293T cells transiently transfected with a Myc-labeled BCAM point mutant, where the structurally predicted surface-exposed charged residues had been replaced with alanine. Expression of all constructs in 293T cells was comparable. Antibody binding was assessed by flow cytometry. The results of these experiments are summarized in […]. Figure 6A , Figure 6B and Figure 6C middle.

[0286] Most point mutations have little or no effect on the ability of the tested antibody to bind to human BCAM. Figure 6A It can be seen that the E180A substitution interferes with the binding of antibody 477 to human BCAM, indicating that this residue is part of the epitope recognized by this antibody. Figure 6B The reduced APC signal and broad binding spectrum observed suggest that antibody 452 partially requires charged residues surrounding the laminin-binding domain for binding BCAM. Figure 6C The narrower binding spectrum and higher APC signal observed indicate that the charged residues surrounding the laminin binding domain are not essential for the binding of antibody 929 to human BCAM. These observations further suggest that each of antibodies 452, 477, and 929 binds to a different epitope on the surface of human BCAM.

[0287] Epitope characterization by sequence alignment with mouse BCAM The data presented in Example 1 and Table 6 indicate that antibodies 477 and 929 bind to human and cynomolgus monkey BCAM, but not to mouse BCAM. This suggests that the differences between the residues of human and cynomolgus monkey BCAM and the residues of mouse BCAM are sufficient to prevent the binding of these antibodies, and therefore, one or more of these residues may be involved in the epitope of each antibody.

[0288] To further characterize these residues, the sequences of human BCAM (i.e., residues 32 to 547 of UniProtKB ID: P50895, corresponding to residues 1 to 516 of SEQ ID NO: 1), cynomolgus monkey BCAM (SEQ ID NO: 28, corresponding to residues 32 to 547 of UniProtKB ID: A0A2K5TW87_MACFA), and mouse BCAM (SEQ ID NO: 29, corresponding to residues 26 to 541 of UniProtKB ID: Q9R069) were compared using the MUSCLE alignment tool. Figure 7A As shown.

[0289] In Example 2 and Figure 2B and Figure 2AThe data presented indicate that antibodies 477 and 929 require the human BCAM domain V1-V2 for binding. The following residues were found to be present only in the mouse BCAM within the sequences that constitute the human BCAM: L9, K16, S17, I19, T25, G26, T27, H28, D29, T38, R40, S41, P45, A50, M52, L57, Q58, V59, M61, D63, T64, R65, S68, Q72, L73, Q76, L81, E83, A84, E89. R97, A104, A108, R109, L110, N111, K115, A118, E132, D133, S134, N142, R144, A149, K151, V165, M167, P169, E170, M173, S175, E180, L185, R196, S203, S211, E214, R216, Q237, P223, and H226.

[0290] Characterization of the role of additional point mutations Human BCAM variants were prepared in which surface-exposed charged residues predicted by a single structure, identified by MUSCLE sequence alignment, were replaced with amino acids having characteristics opposite to the native residues. These BCAM point mutants were transiently transfected into 293T cells in Myc-tagged form. Expression of all constructs in 293T cells was generally comparable; however, a certain proportion of untransfected cells were observed in cells transfected with wild-type BCAM. Antibody binding was assessed by flow cytometry. The results of these experiments are summarized in […]. Figure 7B and Figure 7C middle.

[0291] Figure 7B The introduction of R40E into human BCAM resulted in the loss of antibody 929 binding. Conversely, antibody 929 binding to human BCAM was unaffected by the introduction of the S68R or K16E mutations. These data suggest that the R40 residue in human BCAM can form part of the epitope that antibody 929 binds to.

[0292] Figure 7C The results show that the introduction of P223H eliminated the binding of antibody 477 to human BCAM. In contrast, the binding of antibody 477 to human BCAM was not affected by the introduction of the mutations E244K or H226R. These data suggest that the P223 residue of human BCAM can form part of the epitope bound by antibody 477 (possibly identified by alanine substitution in addition to E180).

[0293] Combination with BCAM polymorphism The ability of antibodies 228, 232, 236, 452, 477, 606, 929, and 937 to bind to BCAM polymorphisms was examined. Each antibody was incubated with 293T cells transiently transfected with a construct expressing the BCAM polymorphism. Specifically, the polymorphisms tested were Lu21, Lu20, Lu17, Lu16, Lu13, Lu12, Lu8, Lu6, Lu5, Lu4, and Lu2. Antibody binding was assessed by flow cytometry. Binding to the Lu12 polymorphism was not detected, which may be an artifact due to low expression levels of this construct in 293T cells and / or poor transfection efficiency.

[0294] Antibodies 452 and 477 exhibited similar binding profiles in these experiments, such as Figure 8 As shown in Table 9, both antibodies bind to most of the tested BCAM polymorphisms.

[0295] Table 9. Binding of anti-BCAM antibodies to BCAM polymorphisms.

[0296] Antibody The number of polymorphisms in a combination 228 10 232 10 452 10+ 477 10 606 11 937 8 929 9 236 8 Combining dynamics The binding kinetics of each of antibodies 452, 477, and 929 to human BCAM were determined using biolayer interference (BLI). The BLI curves for each antibody are shown below. Figure 9A , Figure 9B and Figure 9C In the middle. All antibodies have a very slow dissociation rate, which is characteristic of antibody binding. K of each of antibodies 452, 477, and 929 D The values ​​are shown in Table 10.

[0297] Table 10. Binding kinetics of antibodies 452, 477 and 929.

[0298] Antibody Dynamics K D ]]> 452 0.8 nM 477 0.5 nM 929 0.06 nM Example 6. Design and in vitro testing of bispecific T cell connector (BiTE) Two bispecific T-cell connective (BiTE) designs were generated to identify potential adjunctive therapeutic modalities for antibodies 228, 232, 236, 452, 477, 606, and 937. These designs are schematically illustrated in [illustration missing]. Figure 10A and Figure 10B middle.

[0299] Design of anti-BCAM-anti-CD3-T cell connector Single-chain Fvs (scFvs) for antibodies 228, 232, 236, 452, 477, 606, and 937 were constructed. These scFvs were individually fused to anti-CD3 scFvs via adapter sequences, such as... Figure 10AAs shown in Table 11. The ability of the derived bispecific T cell connective (BiTE) shown in Table 11 to activate T cells was tested using the Jurkat-NFAT-Luc reporter cell line. Anti-EpCAM-anti-CD3 T cell connective soritomob was used as a positive control. An irrelevant antibody (A19) was used as a negative control. The results of this experiment are shown in... Figure 11A In this study, BiTE targeting BCAM effectively activated T cells, as indicated by an increase in relative light units (RLU). Notably, BiTE based on antibody 477 and scFv activated T cells at levels comparable to soritosmumab. Similar results were obtained with the BiTE construct based on 452.

[0300] Table 11. BiTE constructs.

[0301] BiTE construct SEQ ID NO sequence 228 BiTE 101 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVAVISPDGIDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRRGYSYGNFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIDKYLSWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGPGTKVDIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 232 BiTE 102 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGDTNYAQEFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARKRDGGGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSRSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVDIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 236BiTE 103 EVQLLESGGGLVQPGGSLRLSCAASGFAFSNDGMHWVRQAPGKGLEWISYISGSSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAAAGTGGWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISTYLSWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPITFGQGTRLEIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 452BiTE 104 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 477BiTE 105 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 606BiTE 106 EVQLLESGGGLVQPGGSLRLSCAASGFTFGDYDMNWVRQAPGKGLEWVSGISRSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTRGYGQLLDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISMYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTKSIPYTFGQGTKLEIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 937BiTE 107 EVQLLESGGGLVQPGGSLRLSCAASGFAFDDYAMHWVRQAPGKGLEWLAVISYDGSHMSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLVWHPPDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVDIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH 929BiTE 108 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTV SSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIKGG GGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGT STGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKHHHHHH Example 7. Selection Criteria The suitability of antibodies 228, 232, 236, 452, 477, 606, 929, and 937 as therapeutic antibodies was evaluated. Selection criteria are summarized in Tables 12, 13, and 14. This evaluation was based on data collected for each antibody as described in the foregoing examples.

[0302] Table 12. Selection criteria for binding and affinity.

[0303] If the antibody shows binding to BCAM knockout cells of less than 2.5%, and when measured by ELISA, K D Less than 6 nM and / or K when measured by BLI D An antibody with a molecular weight less than 1 nM is considered suitable for therapeutic use. Antibodies 228, 232, 452, 477, 606, 929, and 937 meet these criteria, as shown in Table 12.

[0304] Table 13. Selection criteria for cross-reactivity and multi-reactivity.

[0305] An antibody is considered suitable for therapeutic use if it can bind to at least nine known BCAM polymorphisms. As summarized in Table 13, antibodies 228, 232, 452, 477, 606, and 929 meet this requirement. Furthermore, antibodies are considered suitable for therapeutic use only if they exhibit no homologous cross-reactivity with RAGE, MCAM, or ALCAM and no multiple reactivity with KLH, LPS, or human insulin. All antibodies 228, 232, 236, 452, 477, 606, 929, and 937 meet these additional selection criteria.

[0306] Next, the physical properties of the antibodies that might make them unsuitable for long-term storage were assessed. Furthermore, potential sequence risk factors that could interfere with the affinity, stability, potency, and / or homogeneity of these antibodies were evaluated, particularly due to “stickiness” resulting from having two or more consecutive positively charged residues (e.g., RR or RKR) and N-linked glycosylation. Selection criteria are summarized in Table 14.

[0307] Table 14. Selection criteria for aggregation, thermal stability and sequence risk factors.

[0308] Antibodies 228, 236, 452, 477, and 606 exhibited minimal aggregation. All Fab antibody fragments are thermally stable at or above 68°C. Antibodies 228 and 232 are not considered further due to the presence of two or more consecutive positively charged residues. Such antibodies can be adapted for therapeutic applications by replacing one or more residues of two or more consecutive positively charged residues with one or more non-positively charged amino acids.

[0309] Antibodies 452, 477, and 929 meet all the requirements listed in Tables 12, 13, and 14.

[0310] Example 8. In vitro and in vivo testing of BCAM-based BiTE constructs Further experiments were conducted using a 477-based BiTE construct. This BiTE construct was incubated with human peripheral blood mononuclear cells (PBMCs) obtained from four different donors (PBMC 409, PBMC 627, PBMC 877, and PBMC 905). Various concentrations of the 477-based BiTE construct were tested in this assay. Both T cell activation and cell lysis were measured. The results of these experiments are shown below. Figure 11B and Figure 11C The 477-based BiTE construct effectively activated T cells even at picomolar concentrations (see [link]). Figure 11B It also induces potent target cell toxicity at sub-nanomolar concentrations (see...). Figure 11C ).

[0311] T-cell adaptor design with extended half-life To prolong its half-life in circulation, 477-based BiTE was fused with LALA-PG effector-inactivated IgG1-Fc, such as... Figure 10B As shown. Furthermore, mutations reducing the binding affinity of Fc-protein A are incorporated into the design to allow for more direct purification (e.g., Tustian et al.). Mabs.(As described in 2016;8(4):828-38). The resulting modified BiTE-Fc construct shown in Table 15 binds BCAM with an affinity of approximately 3.5 nM, which is comparable to the conventional hIgG1 format described in the previous section.

[0312] Table 15. Modified BiTE-Fc constructs.

[0313] Modified BiTE-Fc construct SEQ ID NO sequence A 477-based anti-BCAM-BiTE-Fc containing human IgG1 Fc, comprising LALA-PGH435R Y436F 109 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKGGGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG Anti-PSMA-Fc-BiTEe, which has a human IgG1 Fc and comprises LALA-PG H435R Y436F 110 Question KSLIYSASYVYWDVPSRFSGSASGTDFTLTISSVQSEDFATYYCQQYDQQLITFGCGTKLEIKGGGGSDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLDYWGQG TTVTVSSGEGTSTGSGGSGGSGGADDIVLTQSPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKAPKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLEAEDAATYYCQQWSSNPLTFGGGTKVEIKGGGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG The ability of the BiTE-Fc construct to induce T cell activation and target cell lysis in a BCAM-dependent manner was tested. The corresponding BiTE-Fc construct, in which anti-BCAM scFv was replaced with anti-PSMA scFv as a negative control, is also shown in Table 15.

[0314] In the initial experiments, the anti-BCAM BiTE-Fc construct and the control were co-cultured with BCAM-expressing T cell lines at various concentrations. 低 The SKOV3 cell line was incubated together. The percentage of activated T cells observed in this experiment did not exceed 60% (see [link to experiment]). Figure 12A Despite low BCAM expression levels, target cell lysis was induced at picomolar concentrations of the anti-BCAM BiTE-Fc construct (see [link]). Figure 12B The experiment was then repeated using SKOV3 cell lines modified to express recombinant BCAM on their cell surface. This method of expressing BCAM... 高 The cell line had over 80% activated T cells (see [reference]). Figure 12C Furthermore, effective target cell lysis has been observed at picomolar concentrations of the anti-BCAM BiTE-Fc construct (see [link]). Figure 12D No T cell activation or cell lysis was observed with the control construct (i.e., the anti-PSMA BiTE-Fc construct). These results confirm that T cell activation and targeted cell lysis are BCAM expression-dependent. These results also confirm that T cell activation and targeted cell lysis are specific for the anti-BCAM BiTE-Fc construct.

[0315] The experiment was then repeated using the OVSAHO cell line. BCAM expression in this cell line fell between levels observed in wild-type SKOV3 cells and SKOV3 cells modified to express human BCAM. As expected, the observed levels of T cell activation and cell lysis also fell between levels observed in both SKOV3 cell lines. The percentage of activated T cells peaked at approximately 60% (see [link to relevant documentation]). Figure 13A ), and targeted cell lysis was detected in the BiTE-Fc construct at picomolar concentrations (see Figure 13B ).

[0316] The in vivo efficacy of the BCAM-targeting BiTE-Fc construct was evaluated in a mouse tumor model. SKOV3 cells modified to recombinantly express human BCAM were transplanted into immunodeficient NOD2 cells. scid In γ (NSG) mice. Once a tumor is established (i.e., when the tumor size is ≥200 mm). 3 Tumor size (day 0), transplanted human PBMCs. Treatment with the BCAM-targeting BiTE-Fc construct at a dose of 500 µg / kg was initiated on day 1 post-implantation and repeated twice at the same dose, once on day 3 and once on day 5 post-implantation. Tumor volume was monitored twice weekly. Once the tumor size reached >2000 mm... 3 Mice were then euthanized. Mice treated with BiTE-Fc targeting PMSA served as negative controls. Mice treated with PBMCs alone and mice treated with the medium alone (PBS) served as additional controls. Each group consisted of 10 mice.

[0317] The results of this experiment are summarized in Figure 14 Statistically significant differences were observed between mice treated with BCAM-BiTE-Fc and mice treated with PMSA-BiTE-Fc (negative control). Figure 14 It can be seen that the BiTE-Fc construct targeting BCAM is very effective in preventing further tumor growth, while treatment with PMSA-BiTE-Fc has no effect on preventing tumor growth (the tumors in mice in the PMSA-BiTE-Fc treatment group became significantly larger over time).

[0318] Example 9. Design and testing of antibody-drug conjugates (ADCs) To determine their suitability in antibody-drug conjugates, the internalization rate of each of antibodies 452, 477, and 929 was assessed using the IncuCyte® internalization assay. Specifically, antibodies 452, 477, and 929 were labeled with human pHrodo pH-sensitive dye and incubated with OVCAR3 cells. Internalization was measured using an IncuCyte® instrument by quantifying the change in red fluorescence intensity over time. Figure 15A As shown, each of antibodies 452, 477, and 929 was internalized into OVCAR3 cells.

[0319] Antibodies 477, 452, and 929 were then crosslinked with drutecan (DXd) via a GGFG (SEQ ID NO: 85) linker using random cysteine ​​conjugation to provide antibody-drug conjugates (ADCs) for testing, similar to the approved anti-Her2-ADC trastuzumab (ENHERTU). Effective internalization of antibodies 452, 477, and 929 was also demonstrated when they were in ADC form. Following receptor-mediated ADC internalization, the GGFG (SEQ ID NO: 85) linker was cleaved in lysosomes. DXd is a highly cell-permeable topoisomerase inhibitor. Effective bystander killing was observed once released from the antibodies. Figure 15B The illustration shows the efficient internalization of the anti-BCAM-DXd-conjugate ("N" stands for "nucleus," and the arrow indicates the internalized labeled antibody). ADCs internalized into the low-pH environment within the cell (i.e., lysosomes and endosomes) are shown as white dots. The antibody internalization characteristics of conjugated and naked antibodies are similar.

[0320] The drug-to-antibody ratio (DAR) was tested at levels 4 and 8. Figure 16 As illustrated, DXd conjugation under DAR4 and DAR8 does not interfere with the antibody's ability to bind to BCAM. Unconjugated (naked) anti-BCAM antibody and anti-RSV ADC were used as positive and negative controls, respectively. Conjugated antibodies maintain similar binding affinity to naked antibodies.

[0321] OV8 cells were incubated with various concentrations of DAR8-based ADCs (antibodies 477, 452, and 929) to determine the decrease in cell viability after ADC internalization. Anti-RSV ADC was used as a negative control. Figure 17 As shown, all three ADCs were of comparable efficacy. ADCs containing antibodies 477 and 452, respectively, were slightly more effective at sub-nanomolar concentrations. Experiments were repeated with each ADC at DAR4 and DAR8, with a matched anti-RSV ADC serving as a negative control. Results are summarized in […]. Figure 18A (Antibody 929) Figure 18B (Antibody 452) and Figure 18C (Antibody 477). No difference in cytotoxicity was observed between DAR4 and DAR8.

[0322] Next, the anti-BCAM-DXd ADC was tested in vitro in several human BCAM-expressing ovarian (OVSAHO and Kuramochchi) and endometrial (MFE-280) cancer cell lines to assess cytotoxicity. This ADC contained antibody 477. The ability of the ADC to induce cytotoxicity varied significantly among these cell lines. While sub-nanomolar concentrations of the ADC effectively reduced the viability of OVSAHO (see [link to relevant documentation]). Figure 19AHowever, in Kuramochi, only nanomolar concentrations are effective (see...). Figure 19B This is reflected in the IC50 values. The IC50 for OVSAHO is 0.06 nM, while the IC50 for Kuramochi is 2.26 nM. The IC50 of the human ovarian cancer cell line OVCAR3 at 0.11 nM falls in the middle range between OVSAHO and Kuramochi (see [link to relevant documentation]). Figure 19C A minimum IC50 of 0.02 nM was observed in the OVCAR8 cell line modified to recombinantly express human BCAM (see [link]). Figure 19D This is the same IC50 value measured for the human endometrial cancer cell line MFE-280 (see [link]). Figure 19E The IC50 values ​​for each tested cancer cell line are summarized in Table 16 below.

[0323] Table 16. IC50 of anti-BCAM-DXd ADC in various cancer cell lines.

[0324] cell lines IC50 (nM) OVSAHO 0.06 OVCAR8-BCAM 0.02 OVCAR3 0.11 MFE-280 0.02 Kuramochi 2.26 Evaluation of the in vivo efficacy of BCAM-targeting ADCs in mouse tumor models. Immunodeficient NOD cells were transplanted using the human ovarian cancer cell line OVCAR3. scid γ (NSG) mice. Tumors were observed at approximately 200 mm in size. 3 After size establishment (day 0), mice were administered ADC twice (on days 0 and 7) at a dose of 4 mg / kg. The medium alone (PBS) was used. Figure 20 (referred to as "mediator control") or using anti-RSV ADC (in) Figure 20 Mice treated with a method known as “RSV-ADC” served as negative controls. Each treatment group consisted of 10 mice. Tumor volume was monitored twice weekly. When the tumor size exceeded 1000 mm... 3 At that time, the mice were euthanized.

[0325] Figure 20 The results of the above study up to day 56 are shown when anti-BCAM antibody 477 (referred to as “BCAM-ADC” in the figure) conjugated with DXd at a drug:antibody ratio of 4.

[0326] By day 28, nine out of ten mice treated with the BCAM-targeted ADC (“BCAM-ADC”) showed a complete response, i.e., no measurable tumor, and the other mouse showed a partial response, i.e., a tumor volume reduction of >75% to 4 mm. 3 By day 56, mice that had shown a partial response on day 28 also showed a complete response. In contrast, at any time point in the study, mice treated with the control ADC or the mediator alone did not show either a partial or complete response.

[0327] The ADC targeting BCAM alone resulted in a sustained reduction in tumor volume throughout the study. Treatment with the anti-RSV ADC resulted in some initial reduction in tumor volume (presumably due to the nonspecific effect of DXd). However, this reduction was not sustained, and the mean tumor volume subsequently increased rapidly.

[0328] This embodiment demonstrates that an ADC containing the anti-BCAM antibody described herein can effectively reduce the volume of human ovarian cancer tumors. This embodiment also demonstrates that this reduction in tumor volume is sustained.

[0329] Example 10. Design of dual epitope antibodies This embodiment illustrates the preparation of biepithelial anti-BCAM antibodies, each of which comprises a first antibody of the IgG1 type and a second antibody of the scFv type.

[0330] In each case, scFv is fused with the HC of an IgG1 antibody via a linker, thereby providing the “anti-BCAM HC-anti-BCAM scFv” construct. The anti-BCAM HC-anti-BCAM scFv construct, along with anti-BCAM LC, is expressed in mammalian cells to form biepisode antibodies. Three biepisode anti-BCAM antibodies were designed, and their amino acid sequences are listed in Table 17.

[0331] The first biepisode anti-BCAM antibody comprises (i) a 929-based HC (“929HC-477scFv”; SEQ ID NO: 112) fused to a 477-based scFv construct and (ii) a 929-based LC (SEQ ID NO: 65). Except for the C-terminal K residue, the 929-based HC comprises the 929-based VH sequence of SEQ ID NO: 16 and human IgG1 Fc of SEQ ID NO: 76. The 477-based scFv comprises the 477-based VH sequence of SEQ ID NO: 14 and the 477-based VL sequence of SEQ ID NO: 15. The self-assembled biepisode anti-BCAM antibody “929-477scFv” was obtained by co-expressing SEQ ID NO: 112 and SEQ ID NO: 65 in Expi293 cells.

[0332] The second biepisode anti-BCAM antibody comprises (i) a 452-based HC (“452HC-477scFv”; SEQ ID NO: 113) fused to a 477-based scFv construct and (ii) a 452-based LC (SEQ ID NO: 51). Except for the C-terminal K residue, the 452-based HC comprises the 452-based VH sequence of SEQ ID NO: 24 and human IgG1 Fc of SEQ ID NO: 76. The 477-based scFv comprises the 477-based VH sequence of SEQ ID NO: 14 and the 477-based VL sequence of SEQ ID NO: 15. The 452-based LC is also included. The self-assembled biepisode anti-BCAM antibody “452-477scFv” was obtained by co-expressing SEQ ID NO: 113 and SEQ ID NO: 51 in Expi293 cells.

[0333] The third biepisode anti-BCAM antibody comprises (i) a 452-based HC and a 929-based scFv construct (“452HC-929scFv”; SEQ ID NO: 114) and (ii) a 452-based LC (SEQ ID NO: 51). Except for the C-terminal K residue, the 452-based HC comprises the 452-based VH sequence of SEQ ID NO: 24 and the human IgG1 Fc of SEQ ID NO: 76. The 929-based scFv comprises the 929-based VH sequence of SEQ ID NO: 16 and the 929-based VL sequence of SEQ ID NO: 17. The self-assembled biepisode anti-BCAM antibody “452-929scFv” was obtained by co-expressing SEQ ID NO: 114 and SEQ ID NO: 51 in Expi293 cells.

[0334] The signal peptide of the construct used to express the components encoding the aforementioned self-assembled biepisotope anti-BCAM antibody in mammalian cells is cleaved so that the antibody secreted in the supernatant does not contain the signal peptide.

[0335] Table 17. Amino acid sequences of exemplary anti-BCAM HC-anti-BCAM scFV constructs Dual epitope construct SEQ ID NO sequence* 929HC-477scFv 112 mgwsciilflvatatgvhsQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPK FQGRVTMTRDTSTSTVYMELSSLRSEDTAVYCARGIYGHFDYWGQGTLVTVSSASGGGGSGGGGSGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIKR 452HC-477scFv 113 mgwsciilflvatatgvhsQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIKR 452HC-929scFv 114 mgwsciilflvatatgvhsQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSSASGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIKR *The amino acids presented in lowercase letters in the table correspond to the signal peptide presented in SEQ ID NO: 100.

[0336] Example 11. Design and testing of a dual-epitope anti-BCAM ADC This embodiment demonstrates that, compared with single-epitope anti-BCAM ADCs, dual-epitope anti-BCAM ADCs exhibit increased efficacy.

[0337] Using standard procedures, each of the biepithelial anti-BCAM antibodies 929-477scFv, 452-477scFv, and 452-929scFv designed in Example 10 was purified from Expi 293 supernatant using protein A beads. To generate the biepithelial anti-BCAM ADC, the purified biepithelial antibodies were then incubated with the "oYo-Vc anti-microtubule agent monomethylaurestatin E (MMAE)" (ALPHATHERA) linker according to the manufacturer's instructions and photocrosslinked using an ALPHATHERA crosslinking device, see, for example, Hui et al., 2015 Bioconjugate Chemistry 26(8). Crosslinking was verified by SDS-PAGE. A representative schematic diagram of the biepithelial anti-BCAM ADC is shown in [illustration missing]. Figure 21 In the following text, the dual-tablet anti-BCAM ADCs are referred to as “929-477scFvADC”, “452-477scFv ADC”, and “452-929scFv ADC”, respectively.

[0338] The cytotoxicity of each biepisode anti-BCAM ADC was tested in vitro. Specifically, OVCAR3 cells were incubated for 72–96 hours with various concentrations of 929–477scFv ADC, 452–477scFv ADC, or 452–929scFv ADC, or their corresponding control constructs and negative controls, and cell viability was measured using a Cell titer glow assay according to the manufacturer’s instructions. Controls were: (i) “BCAM-IgG-oYo (1)”, a full-length antibody based on 452 or 929 crosslinked with MMAE, corresponding to IgG1 in the biepisode antibody; (ii) “BCAM-IgG-oYo (2)”, a full-length antibody based on 477 or 929 crosslinked with MMAE, corresponding to scFv in the biepisode antibody; and (iii) “IgG-oYo”, an IgG control antibody crosslinked with MMAE (negative control). The results of these experiments are shown in [Table / Table / Insert Table ... Figure 22A , Figure 22B and Figure 22C The IC50 value for each antibody was calculated using the results, as shown in Table 18.

[0339] Table 18. IC50 values of dual-epitope anti-BCAM ADCs and their mono-epitope controls Dual-epitope construct BCAM-IgG-oYo (1) IC 50 (nM) BCAM-IgG-oYo (2) IC 50 (nM) Bisepitope oYoIC 50 (nM) 929-477scFv ADC 0.7 0.4 0.1 452-477scFv ADC 3 0.4 0.08 452-929scFv ADC 3 0.7 0.1 As shown in Table 18, the biepisode formulation improved the cytotoxicity of each anti-BCAM ADC. Surprisingly, each tested biepisode anti-BCAM ADC had an IC50 of approximately 0.1 nM or less, which is a significant improvement compared to the monoepisode control antibody.

[0340] This embodiment demonstrates that biepisode anti-BCAM ADCs are more effective than monoepisode anti-BCAM ADCs in reducing the viability of ovarian cancer cells. The results of this embodiment indicate the efficacy of such biepisode antibodies in cancer therapy.

[0341] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials may be used in the practice or testing of this disclosure, suitable methods and materials are described herein.

[0342] While many embodiments are described herein, changes may be made to this disclosure and examples to provide other methods and compositions. Therefore, it should be understood that the scope is defined by the appended claims in addition to the specific embodiments illustrated by example.

Claims

1. An antibody that specifically binds to human basal cell adhesion molecule (BCAM), wherein the antibody comprises a variable region and a constant region, wherein the variable region comprises a framework region and a complementary determining component for binding human BCAM.

2. An antibody that specifically binds to human basal cell adhesion molecule (BCAM), said antibody comprising: a. Heavy chain complementarity-determining regions (HCDRs) 1 containing the amino acid sequence DYYVH (SEQ ID NO: 2), HCDR 2 containing the amino acid sequence IINPFGGSATYAPKFQG (SEQ ID NO: 3), and HCDR 3 containing the amino acid sequence GIYGHFDY (SEQ ID NO: 4); and b. The light chain complementarity-determining region (LCDR)1 containing the amino acid sequence KSSQSVLSASNNQNYLA (SEQ ID NO: 5), the LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 6), and the LCDR3 containing the amino acid sequence QQYYSSPYT (SEQ ID NO: 7).

3. The antibody of claim 2, wherein the antibody comprises: a. An immunoglobulin heavy chain variable (V) containing at least about 85% identical or identical amino acid sequence to or the same as QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14). H ) area, and b. An immunoglobulin light chain variable (V) containing at least about 85% identical or identical amino acid sequence to or the same as that of DIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 15). L )district.

4. The antibody of claim 2 or 3, wherein the antibody is a single-chain variable fragment (scFv) comprising a sequence having at least about 85% identity with or identical to the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSSGGGGS GGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSASNNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK (SEQ ID NO: 78).

5. An antibody that specifically binds to human basal cell adhesion molecule (BCAM), said antibody comprising: a. HCDR1 containing the amino acid sequence SYAFS (SEQ ID NO: 8), HCDR2 containing the amino acid sequence GIIPFSGTTNYAQKFQG (SEQ ID NO: 9), and HCDR3 containing the amino acid sequence DPILSFWSGYYYYYGMDV (SEQ ID NO: 10); and b. LCDR1 containing the amino acid sequence RASQSISSWLA (SEQ ID NO: 11), LCDR2 containing the amino acid sequence DASNLET (SEQ ID NO: 12), and LCDR3 containing the amino acid sequence QQSYSTPPT (SEQ ID NO: 13).

6. The antibody of claim 5, wherein the antibody comprises: a. Contains an immunoglobulin heavy chain variable (VH) region containing at least about 85% identical or identical amino acid sequence to or the same as that of QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO:16), and b. Contains an immunoglobulin light chain variable (VL) region containing at least about 85% identical or identical amino acid sequences to or the same as those of DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

7. The antibody of claim 5 or 6, wherein the antibody is a single-chain variable fragment (scFv) comprising a sequence having at least about 85% identity with or identical to the following sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVT VSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 79).

8. An antibody that specifically binds to human basal cell adhesion molecule (BCAM) and competes with antibodies comprising: a. Variable immunoglobulin heavy chain (V H The region contains the amino acid sequence. QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYVHWVRQAPGQGLEWMGIINPFGGSATYAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGIYGHFDYWGQGTLVTVSS (SEQ ID NO: 14), and Immunoglobulin light chain variable (V L The region contains the amino acid sequence. OR b. Variable (VH) region of immunoglobulin heavy chain, which contains an amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPFSGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTDPILSFWSGYYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16), and Immunoglobulin light chain variable (V L The region contains the amino acid sequence. DIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTRLEIK (SEQ ID NO: 17).

9. The antibody according to any one of claims 2 to 8, wherein the antibody is capable of inhibiting the binding of human BCAM to laminin α-5.

10. An antibody that specifically binds to human basal cell adhesion molecule (BCAM), said antibody comprising: a. HCDR1 containing the amino acid sequence SYYMH (SEQ ID NO: 18), HCDR2 containing the amino acid sequence WMNPNSGDSGYAQKFQG (SEQ ID NO: 19), and HCDR3 containing the amino acid sequence TYSSSWIFDY (SEQ ID NO: 20); and b. LCDR1 containing the amino acid sequence KSSQSVLYSSNNKNYLA (SEQ ID NO: 21), LCDR2 containing the amino acid sequence WASTRES (SEQ ID NO: 22), and LCDR3 containing the amino acid sequence QQYYSIPIT (SEQ ID NO: 23).

11. The antibody of claim 10, wherein the antibody comprises: a. An immunoglobulin heavy chain variable (V) containing at least about 85% identical or identical amino acid sequence to or the same as QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO: 24). H ) area, and b. An immunoglobulin light chain variable (V) containing at least about 85% identical or identical amino acid sequence to or the same as that of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25). L )district.

12. The antibody of claim 10 or 11, wherein the antibody is a single-chain variable fragment (scFv) comprising a sequence having at least about 85% identity with or identical to the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 82).

13. An antibody that specifically binds to human basal cell adhesion molecule (BCAM) and competes with antibodies comprising: a. A variable immunoglobulin heavy chain containing the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNSGDSGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASTYSSSWIFDYWGQGTLVTVSS (SEQ ID NO:24) H ) area, and b. Variable immunoglobulin light chain containing the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPITFGQGTRLEIK (SEQ ID NO: 25) L )district.

14. The antibody according to any one of claims 10 to 13, wherein the antibody is capable of increasing the binding of human BCAM to laminin α-5.

15. The antibody as claimed in any of the preceding claims, wherein the antibody has a dissociation constant (K0) of less than about 10 nM or less than about 1 nM. D (By combining human BCAM, as determined using cell-based assays.) 16. The antibody of claim 15, wherein the K D It ranges from approximately 0.1 nM to approximately 5 nM, as determined using cell-based assays.

17. The antibody as claimed in any of the preceding claims, wherein the antibody is human or humanized.

18. The antibody as claimed in any of the preceding claims, wherein the antibody is selected from: a. IgG antibodies, such as IgG1 or IgG3 antibodies; b. Bispecific or multispecific antibodies; c. Single-chain antibodies; or d. Double antibody.

19. The antibody of claim 18, wherein the antibody comprises or comprises a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, and a single-chain variable fragment (scFv).

20. The antibody as claimed in any of the preceding claims, wherein the antibody comprises an Fc region.

21. The antibody of claim 20, wherein the Fc region comprises an LALA mutation.

22. The antibody of claim 21, wherein the Fc region further comprises a PG mutation.

23. The antibody of any one of claims 1 to 22, wherein the antibody further comprises an antibody component for binding CD3, such as anti-CD3 scFv.

24. The antibody of any one of claims 1 to 23, wherein the antibody is conjugated with a drug.

25. The antibody of claim 24, wherein the drug is an anticancer drug.

26. The antibody of claim 25, wherein the anticancer drug is a cytotoxic agent or an immunomodulator.

27. The antibody of claim 26, wherein the cytotoxic agent is a microtubule inhibitor or a DNA damaging agent.

28. The antibody of claim 27, wherein the microtubule inhibitor comprises aureatin, maytansine alkaloids, or tubulolysin.

29. The antibody of claim 28, wherein the DNA damaging agent is an agent that induces DNA double-strand breaks, a DNA alkylating agent, a topoisomerase inhibitor, or a DNA cross-linking agent.

30. The antibody of claim 29, wherein the agent that induces DNA double-strand breaks is calichiomycin.

31. The antibody of claim 29, wherein the DNA alkylating agent is docalomycin.

32. The antibody of claim 29, wherein the topoisomerase inhibitor is eczema.

33. The antibody of claim 29, wherein the DNA cross-linking agent is pyrrolobenzodiazepine.

34. The antibody of claim 26, wherein the immunomodulator is a TLR agonist or a STING agonist.

35. The antibody of any one of claims 24 to 34, wherein the drug is conjugated to the antibody via a cleavable linker.

36. The antibody of claim 35, wherein the cleavable adapter comprises a protease cleavage site.

37. The antibody of claim 36, wherein the protease is a lysosomal enzyme.

38. The antibody of any one of claims 24 to 34, wherein the drug is conjugated to the antibody via an indestructible linker.

39. A dual epitope antibody comprising at least one antibody according to any one of the preceding claims.

40. A bispecific T-cell connector (BiTE), said bispecific T-cell connector (BiTE) comprising a. An antibody component for binding human basal cell adhesion molecule (BCAM); b. Antibody components for binding CD3; c. Connecting the antibody component for binding human BCAM to a connector of the antibody component for binding CD3; and optionally... d. Components used to extend half-life.

41. The BiTE of claim 40, wherein the antibody component comprises the antibody of any one of claims 1 to 22.

42. A pharmaceutical composition comprising an antibody that specifically binds to human basal cell adhesion molecule (BCAM), the antibody comprising a variable region and a constant region, wherein the variable region comprises a framework region and a complementary determining element for binding human BCAM, the antibody being mixed with a pharmaceutically acceptable carrier.

43. A pharmaceutical composition comprising an antibody as described in any one of claims 1 to 38, a biepisode antibody as described in claim 39, or a BiTE as described in claim 40 or 41, and one or more pharmaceutically acceptable excipients.

44. A method of treating cancer in a human subject, the method comprising administering to the human subject an effective amount of an antibody that specifically binds to human basal cell adhesion molecule (BCAM), the antibody comprising a variable region and a constant region, wherein the variable region comprises a framework region and a complementary determining component for binding human BCAM, the antibody being mixed with a pharmaceutically acceptable carrier.

45. A method of treating cancer in a subject in need, wherein the method comprises administering to the subject a therapeutically effective amount of an antibody as described in any one of claims 1 to 38, a biepisode antibody as described in claim 39, a BiTE as described in claim 40 or 41, or a pharmaceutical composition as described in claim 43.

46. ​​The method of claim 45, wherein the cancer is ovarian cancer or endometrial cancer.

47. The method of claim 46, wherein the ovarian cancer is high-grade ovarian cancer.

48. The method of claim 46 or 47, wherein the ovarian cancer is serous ovarian cancer.

49. The method of any one of claims 45 to 48, wherein the subject is untreated.

50. The method of any one of claims 45 to 48, wherein the subject is treated with one or more other therapies.

51. The method of claim 50, wherein the one or more additional therapies include surgery and / or chemotherapy.

52. A primary nucleic acid and a secondary nucleic acid, wherein: a. The first nucleic acid comprises an immunoglobulin heavy chain variable (V) encoding an antibody as described in any one of claims 4, 8, or 13. H The nucleotide sequence of the region, and b. The second nucleic acid contains a variable (V) light chain of an immunoglobulin encoding the same antibody. L The nucleotide sequence of the region.

53. A first expression vector and a second expression vector, wherein the first expression vector and the second expression vector respectively comprise the first nucleic acid and the second nucleic acid as described in claim 52.

54. A nucleic acid that encodes an antibody as claimed in any one of claims 1 to 25.

55. An expression vector comprising the nucleic acid as described in claim 54.

56. A host cell comprising the expression vector as described in claim 53 or 55.

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