Anti-ABCC1 antibodies and uses thereof

Abstract

Antibodies that target the cellular efflux pump ABCC1 are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of ABCC1 expression, the level of ABCC1 expression, and / or to inhibit ABCC1 function in cells, e.g., tumor cells, are also disclosed. Methods for treating a subject for cancer are also provided, comprising administering to the subject an anti-ABCC1 antibody disclosed herein. The subject antibody can be a bispecific antibody. The bispecific antibody can bind to ABCC1 and a tumor-associated antigen (TAA).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 073826, filed September 2, 2020, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listings provided as text files The Sequence Listing is provided herein as a text file "KNJY-006WO SEQ LIST_ST25.txt" having a size of 151 KB, created on August 24, 2021. The contents of the text file are incorporated herein by reference in their entirety.

[0003] Introduction Drug resistance, a well-known phenomenon that occurs when a disease becomes resistant to pharmaceutical treatment, is a major and growing problem in various fields of medicine, including oncology. Many types of cancer are initially sensitive to chemotherapy, but over time, they can develop resistance through these and other mechanisms, including DNA mutations and metabolic changes that promote drug inhibition, degradation, and enhanced excretion.

[0004] Efflux pumps (EPs) are proteins expressed by living cells that have evolved to naturally efflux various compounds from the cell. Members of the ATP-binding cassette (ABC) transporter family of proteins are examples of EPs that enable drug efflux. While transporter structure varies from protein to protein (e.g., there are 49 known ABC family members in humans), they are all classified by the presence of two distinct domains: a highly conserved nucleotide-binding domain and a more variable transmembrane domain. Multidrug resistance protein 1 (MDR1), encoded by the ATP-binding cassette subfamily B member 1 (ABCB1) gene, was the first of these to be identified and has been extensively studied. ATP-binding cassette subfamily C member 1 (ABCC1) expression increases in response to treatment with certain chemotherapeutic agents.

[0005] EP allows tumors to develop resistance to chemotherapeutic agents. Such resistance is often associated with enhanced efflux of chemotherapeutic agents from drug-resistant cells. When applied to two or more chemotherapeutic agents, this resistance is called multidrug resistance (MDR).

[0006] Thus, there is a need to develop reagents that can be used to assay for the expression of EP and / or to inhibit EP. Summary of the Invention

[0007] Antibodies that target the cellular efflux pump ATP-binding cassette subfamily C member 1 (ABCC1) are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of ABCC1 expression, the level of ABCC1 expression, and / or to inhibit ABCC1 function in cells, e.g., tumor cells, are also disclosed. Methods for treating a subject for cancer, comprising administering to the subject an anti-ABCC1 antibody disclosed herein, are also provided. [Brief explanation of the drawings]

[0008] [Figure 1]

[0023] Figure 1 shows the results of titration binding of the indicated anti-ABCC1 monoclonal antibodies to the doxorubicin-resistant lung cancer cell line H69AR (ATCC® CRL-11351), which endogenously expresses ABCC1. H69AR (ATCC CRL-11351) was established from NCI-H69 cells grown in the presence of increasing concentrations of adriamycin (doxorubicin) for a total of 14 months. [Figure 2] 2A-2B show the results of titration binding of the indicated anti-ABCC1 monoclonal antibodies to human and cynomolgus ABCC1-overexpressing rat C6 glioma cell lines. [Figure 3] Figures 3A-3C, 4, 5A-5B, and 6A-6B show the results of titrations of additional anti-ABCC1 monoclonal antibodies to human and cynomolgus monkey ABCC1-overexpressing rat C6 glioma cell lines. "Second Ab only" refers to the absence of primary antibody addition prior to binding with the secondary antibody (i.e., second Ab) to provide a negative control. [Figure 4] Figures 3A-3C, 4, 5A-5B, and 6A-6B show the results of titrations of additional anti-ABCC1 monoclonal antibodies to human and cynomolgus monkey ABCC1-overexpressing rat C6 glioma cell lines. "Second Ab only" refers to the absence of primary antibody addition prior to binding with the secondary antibody (i.e., second Ab) to provide a negative control. [Figure 5] Figures 3A-3C, 4, 5A-5B, and 6A-6B show the results of titrations of additional anti-ABCC1 monoclonal antibodies to human and cynomolgus monkey ABCC1-overexpressing rat C6 glioma cell lines. "Second Ab only" refers to the absence of primary antibody addition prior to binding with the secondary antibody (i.e., second Ab) to provide a negative control. [Figure 6]Figures 3A-3C, 4, 5A-5B, and 6A-6B show the results of titrations of additional anti-ABCC1 monoclonal antibodies to human and cynomolgus monkey ABCC1-overexpressing rat C6 glioma cell lines. "Second Ab only" refers to the absence of primary antibody addition prior to binding with the secondary antibody (i.e., second Ab) to provide a negative control. [Figure 7A] 7A-7B show the results of an ABCC1 shedding assay performed using HEK293T cells expressing human ABCC1. [Figure 8] 8A-8C present the titration binding and efflux assay characterization of humanized anti-ABCC1 monoclonal antibodies. [Figure 9] 9A-9C show the binding of humanized anti-ABCC1 monoclonal antibodies to human and cynomolgus ABCC1-overexpressing rat C6 glioma cell lines. [Figure 10] 10A-10B show the binding of various humanized C1.851 anti-ABCC1 antibodies to human and cynomolgus ABCC1-overexpressing rat C6 glioma cell lines. [Figure 11] Figures 11A-11C show the binding of four humanized C1 / KT9 bispecific antibodies to human and cynomolgus monkey C6 cell lines overexpressing ABCC1 and KT9, respectively. Schematic bispecific antibody structures are also shown. KT9 represents the anti-PD-L1 monoclonal antibody atezolizumab. The bispecific antibodies contain the heavy and light chains from the indicated ABCC1 antibody and the scFv region formed from the KT9 antibody. [Figure 12] Figures 12A-12C show the binding of four humanized C1 / KT1 bispecific antibodies to 293T cells expressing human ABCC1 and 293T cells expressing human or cynomolgus monkey KT1, respectively. KT1 represents the anti-ErbB2 (anti-HER2) monoclonal antibody trastuzumab. The bispecific antibodies contain heavy and light chains from the indicated anti-ABCC1 antibodies and an scFv region formed from the KT1 antibody. [Figure 13]13A-13B, 14A-14B, and 15 show the effect of tested anti-ABCC1 monoclonal antibodies on vincristine cytotoxicity in a 293T cytotoxicity assay. [Figure 14] 13A-13B, 14A-14B, and 15 show the effect of tested anti-ABCC1 monoclonal antibodies on vincristine cytotoxicity in a 293T cytotoxicity assay. [Figure 15] 13A-13B, 14A-14B, and 15 show the effect of tested anti-ABCC1 monoclonal antibodies on vincristine cytotoxicity in a 293T cytotoxicity assay. [Figure 16] Three tested anti-ABCC1 monoclonal antibodies inhibit tumor growth in vivo in the H69AR cytotoxicity assay. The assay evaluates the effect of the tested antibodies on adriamycin cytotoxicity of the H69AR cell line, an adriamycin-selected C1-positive variant of the human small cell lung cancer cell line, NCI-H69. Tumors formed from the H69AR cell line are resistant to adriamycin (doxorubicin). All three of the tested anti-ABCC1 monoclonal antibodies sensitized tumors to adriamycin. [Figure 17] We demonstrate that anti-ABCC1 monoclonal antibodies C1-831 and C1-737A inhibit tumor growth in vivo in the CT26 syngeneic mouse tumor model, and that tumor growth inhibition by these antibodies is enhanced by doxorubicin.

[0009] definition The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, Fd, Fab', Fv, and F(ab')2, chimeric antibodies, humanized antibodies, monoclonal antibodies, single-chain antibodies including antibodies containing only a heavy chain (e.g., VHH camelid antibodies), bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, or the like. Antibodies may be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair). Antibodies may also be bound to a solid support, including, but not limited to, a polystyrene plate or beads. Antibodies may be monovalent or bivalent. Antibodies may be conjugated to a toxic moiety, such as a chemotherapeutic agent.

[0010] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules including antibodies containing only heavy chains (e.g., VHH camelid antibodies), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0011] An "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site comprising the three CDRs from each variable domain.

[0012] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0013] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0014] "Single-chain Fv," "sFv," or "scFv" antibody fragments are fragments of the V of an antibody. H and V LIn some embodiments, an Fv polypeptide comprises a V domain, and these domains are present in a single polypeptide chain. H Domains and V L The sFv further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0015] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to form two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0016] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, expressed as the dissociation constant (Kd). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0017] The term "binding" refers to a direct association between two molecules by ionic and / or hydrogen bonding interactions, including covalent interactions, electrostatic interactions, hydrophobic interactions, and interactions such as salt bridges and water bridges. ABCC1-specific antibodies specifically bind to an epitope within the ABCC1 polypeptide. The epitope can be a linear epitope formed by a contiguous stretch of amino acids, or a nonlinear or conformational epitope formed by a non-contiguous stretch of amino acids. Non-specific binding occurs at approximately 10 -7 Binding with an affinity of less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 This refers to binding based on affinity, such as M.

[0018] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are hypervariable regions interspersed with more conserved regions called "framework regions (FR)." CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definition includes overlapping amino acid residues or subsets thereof when compared against each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues that encompass the CDRs as defined by each of the above-cited documents are set forth below in Table 1 for comparison. [Table 1]

[0019] As used herein, the term "framework," when used in reference to an antibody variable region, is intended to refer to all amino acid residues outside the CDR regions in the variable region of an antibody. The variable region framework is generally a discontinuous amino acid sequence about 100 to 120 amino acids in length, but is intended to refer only to those amino acids outside the CDRs. As used herein, the term "framework region" is intended to refer to each domain of the framework separated by the CDRs. A VH chain can comprise three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, a VL chain can comprise three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The terms VH chain and VH region are used interchangeably herein. The terms VL chain and VL region are used interchangeably herein.

[0020] As used herein, the term "antibody" encompasses a tetramer of two heavy chains and two light chains, where the heavy and light chains are interconnected, for example, by disulfide bonds. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains contain binding regions that interact with antigens. The constant regions of antibodies typically mediate the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system. The term "antibody" includes the immunoglobulin types IgA, IgG, IgE, IgD, IgM, and their subtypes. In some embodiments, the subject antibody is an IgG isotype, e.g., IgG1.

[0021] As used herein, the term "immunoglobulin" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains (approximately 25 kD or 214 amino acids) are encoded by a variable region gene (approximately 110 amino acids) at the N-terminus and a kappa or lambda constant region at the C-terminus. Full-length immunoglobulin heavy chains (approximately 50 kD or 446 amino acids) are encoded by a variable region gene (approximately 116 amino acids) at the N-terminus and one of the other aforementioned constant region genes, e.g., gamma (encoding approximately 330 amino acids), at the C-terminus. In some embodiments, a subject antibody comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0022] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to an antigen. Examples of binding fragments include: (i) a Fab fragment (a monovalent fragment comprising, e.g., consisting of, the VL, VH, CL, and CH1 domains), (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), (iii) a Fd fragment (comprising, e.g., consisting of the VH and CH1 domains), (iv) a Fv fragment (comprising, e.g., consisting of the VH and VL domains of a single arm of an antibody), (v) a dAb fragment (comprising, e.g., consisting of the VH domain), (vi) a dAb fragment (comprising, e.g., consisting of the VH domain), (vii) a dAb fragment (comprising, e.g., consisting of the VH domain), (viii) a dAb fragment (comprising, e.g., consisting of the VH domain), (viiii ... (vi) isolated CDRs; (vii) single-chain Fvs (scFvs) (e.g., comprising, e.g., consisting of, the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant means such that the VH and VL domain pair forms a monovalent molecule); (viii) diabodies (comprising, e.g., consisting of, two scFvs whose VH and VL domains are not paired such that they do not form a monovalent molecule, and wherein the VH of each one of the scFvs pairs with the VL domain of the other scFv to form a bivalent molecule).

[0023] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0024] A "human antibody" is one having an amino acid sequence corresponding to an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0025] A "human consensus framework" is a framework (FR) representing the most commonly occurring amino acid residues in a selection of human immunoglobulin variable light (VL) or variable heavy (VH) framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, Md. (1991), vols. 1-3. In one embodiment, for VL, the subgroup is kappa I as in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0026] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and human framework (FR) regions. At least a portion of the constant region of a humanized antibody is derived from a human antibody, such as a human IgG1 antibody. In a preferred embodiment, the antibody molecule disclosed herein comprises a heavy chain comprising a variable heavy chain region provided herein and a human IgG1 constant region having the amino acid sequence set forth in UniProt:P01857-1, version 1. In a preferred embodiment, the antibody molecule disclosed herein comprises a light chain comprising a variable light chain region provided herein and a human light chain constant region. In a preferred embodiment, the human light chain constant region is a human kappa light chain constant region having the amino acid sequence set forth in UniProtKB / Swiss-Prot:P01834.2. In certain embodiments, the human IgG1 heavy chain constant region present in a subject antibody may comprise mutations, e.g., substitutions that modulate Fc function. For example, LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation can be introduced to reduce antibody-dependent cellular cytotoxicity (ADCC). The numbering of substitutions is based on the EU numbering system. The "EU numbering system" or "EU index" is commonly used when referring to residues in immunoglobulin heavy chain constant regions (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0027] A "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

[0028] The term "epitope" refers to a region of an antigen that is recognized by the immune system, e.g., by an antibody, a B cell, or a T cell. For example, an epitope is the specific region of an antigen to which an antibody binds.

[0029] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 90%, 95%, or 98% by weight of the antibody as determined by the Lowry method, e.g., greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. In some cases, isolated antibodies are prepared by at least one purification step.

[0030] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. "Chemotherapeutic agents," also called "anti-tumor agents," can be cytotoxic agents used to treat cancer or other diseases or disorders.

[0031] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or side effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any treatment of disease in mammals, including humans, and includes (a) preventing the disease from occurring in a subject who is susceptible to the disease but has not been diagnosed as having it, (b) inhibiting the disease, i.e., arresting its development, and (c) palliating the disease, i.e., causing regression of the disease.

[0032] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0033] A "therapeutically effective amount" or "effective amount" refers to the amount of a target-specific antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment in the disease. A "therapeutically effective amount" will vary depending on the antibody, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0034] The term "refractory" as used herein refers to a disease or condition that does not respond to treatment. With respect to cancer, "refractory cancer," as used herein, refers to a cancer that does not respond to treatment. A refractory cancer may be resistant at the start of treatment or may become resistant during treatment. A refractory cancer may also be referred to as a resistant cancer.

[0035] A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition includes blood and other liquid samples from a living body, solid tissue samples such as biopsy specimens, or tissue cultures or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in any way after their procurement, for example, by treatment with reagents, solubilization, or enrichment for particular components such as polynucleotides. The term "biological sample" encompasses clinical samples, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0036] The percent identity between a pair of sequences can be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region, including gaps. Identity scoring counts only perfect matches and does not take into account the degree of similarity of the amino acids to each other. Only internal gaps are included in the length, not gaps at the ends of the sequences. Percent identity = (number of matches x 100) / length of aligned region (with gaps).

[0037] The phrase "conservative amino acid substitution" refers to substitutions of amino acid residues within the following groups: 1) L, I, M, V, F, 2) R, K, 3) F, Y, H, W, R, 4) G, A, T, S, 5) Q, N, and 6) D, E. Conservative amino acid substitutions may preserve the activity of a protein by replacing an amino acid in a protein with an amino acid having a side chain of similar acidity, basicity, charge, polarity, or size.

[0038] Guidance for substitutions, insertions, or deletions can be based on alignment of the amino acid sequences of proteins from different species, or can be from a consensus sequence based on multiple proteins with the same or similar function.

[0039] The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0040] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in conjunction with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region operably linked thereto.

[0041] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex or signaling through the signaling domain of an appropriate NK receptor or CAR. Stimulation can mediate altered expression of specific molecules.

[0042] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signal sequence that regulates immune cell activation in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one embodiment, the signal is a primary signal initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, resulting in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences of particular use in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0043] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0044] The term "autologous" refers to any material derived from the same individual into which it is later reintroduced.

[0045] "Intracellular signaling domain," as used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-containing cell, e.g., a CAR-T cell. Examples of immune effector function, e.g., in a CAR-T cell, include helper activity, including cytolytic activity and cytokine secretion.

[0046] As used herein, "immune effector cells" refers to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes. DETAILED DESCRIPTION OF THE INVENTION

[0047] Antibodies that bind to the cellular efflux pump ABCC1 are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of ABCC1 expression, the level of ABCC1 expression, and / or to inhibit ABCC1 function in cells, e.g., tumor cells, are also disclosed. Methods for treating a subject for cancer, comprising administering to the subject an anti-ABCC1 antibody as disclosed herein, are also provided.

[0048] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, and that the scope of the present invention will be limited only by the appended claims.

[0049] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in the stated range, is encompassed within the invention. The upper and lower limits of these narrower ranges may independently be included in the narrower ranges and are also encompassed within the invention, subject to any specific excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in the invention.

[0050] Certain ranges are described herein by numerical values ​​preceded by the term "about." The term "about" is used herein to provide textual support for the exact number it precedes, as well as for numbers that are near or approximately near the number preceded by the term. In determining whether a number is near or approximately near a specifically recited number, the unrecited number that is near or approximately near may be a number that, in the context in which it is presented, provides substantially the equivalent value of the specifically recited number.

[0051] 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 to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are now described.

[0052] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of its disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0053] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a precedent for the use of such exclusive terminology, such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claimed elements.

[0054] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and characteristics which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0055] Although the methods and compositions have been described or will be described for grammatical fluidity with functional descriptions, it is expressly understood that the claims should not be construed as necessarily limited by "means" or "step" limitation constructions unless expressly formulated under 35 U.S.C. 112(f), but will be given the full scope of meaning and equivalents of the definitions provided by the claims under the judicial doctrine of equivalents, and that if the claims are expressly formulated under 35 U.S.C. 112(f), they will be given the full statutory equivalents under 35 U.S.C. 112(f).

[0056] antibody As summarized above, the present disclosure provides antibodies that bind to the cellular efflux pump ABCC1, which is expressed on the surface of mammalian cells, e.g., human cells. ABCC1, also known as glutathione-S-conjugate-translocating ATPase ABCC1 or multidrug resistance-associated protein 1 (MRP1), is an energy-dependent pump that effluxes drugs and organic anions across the plasma membrane. It contains 17 transmembrane helices connected by extracellular and cytoplasmic loops. ABCC1 mediates resistance to, among other drugs, doxorubicin, etoposide, and vincristine.

[0057] In some embodiments, the antibodies disclosed herein bind to one or more sites on the extracellular domain of ABCC1. In certain embodiments, the anti-ABCC1 antibodies of the present disclosure bind to human ABCC1. In certain embodiments, the anti-ABCC1 antibodies of the present disclosure bind to human ABCC1 expressed on the cell surface of human cells, e.g., cancer cells.

[0058] The antibodies of the present disclosure may have one or more of the following characteristics. i) inhibiting efflux from ABCC1; ii) increasing the sensitivity of cancer cells to treatment with a chemotherapeutic agent, thereby reducing the IC50 of the chemotherapeutic agent by at least two-fold; iii) binds to human and cynomolgus monkey ABCC1 on the cell surface; iv) is effective in an in vitro cell killing assay; v) is effective in inhibiting tumor growth even in the absence of chemotherapy, and vi) has affinity for ABCC1 in a lower range such that it binds to cancer cells that express ABCC1 at higher levels compared to non-cancer cells and binds significantly less to non-cancer cells.

[0059] As used herein, EC50 refers to the concentration of antibody that provides half of the maximal response (e.g., half of the maximal fluorescence intensity). Antibodies of the present disclosure may have an EC50 of 100 nM or less, e.g., 100 nM to 4 nM, 80 nM to 4 nM, 60 nM to 4 nM, 40 nM to 4 nM, 30 nM to 4 nM, 20 nM to 4 nM, 15 nM to 4 nM, or 10 nM to 4 nM. The EC50 of many test antibodies is determined by flow cytometry or ELISA. For example, flow cytometry may involve contacting cells expressing ABCC1 (e.g., human ABCC1) with an antibody in flow cytometry buffer, where the antibody is serially diluted, and incubating at room temperature or 4°C for a period sufficient for the antibody to bind to the cells (e.g., 10 minutes to 1 hour). After incubation, the cells may optionally be washed to remove non-specifically bound antibodies and / or contacted with a fluorescently labeled secondary antibody that specifically binds to the test antibody. After incubation, the fluorescently labeled secondary antibody may be removed and the cells washed. The washed cells may be sorted by flow cytometry, and the number of cells bound to the fluorescently labeled secondary antibody may be counted. The concentration providing half of the maximal response (e.g., half of the maximal fluorescence intensity) is measured as the EC50. In a variation of the flow cytometry assay, the cells may be 293T cells overexpressing ABCC1.

[0060] The IC50 of a test antibody can be determined by measuring inhibition of cell growth. IC50 can be measured using the test antibody alone to determine the concentration of antibody that produced a half-maximal response. The IC50 of a chemotherapeutic agent can be measured in the absence and presence of the test antibody to determine the effect of the antibody on the IC50 chemotherapeutic agent. The chemotherapeutic agent can be doxorubicin, daunorubicin, etoposide, vincristine, etc. The cells can be a cancer cell line. The cancer cell line can be H69AR, a doxorubicin-selected C1-positive variant of the lung cancer cell line, H69. When determining the IC50 of an antibody, the cells can be contacted with the antibody alone, and the antibody is tested in serial dilutions. The cells can be contacted with the antibody and a chemotherapeutic agent to determine the effect of the antibody on the IC50 of the agent, and the agent is tested in serial dilutions. The cells can be incubated at 37°C for a period of time (e.g., 24 to 84 hours), and cell viability can be assessed using standard reagents and methods. The antibodies disclosed herein may sensitize cancer cells to treatment with a chemotherapeutic agent, thereby reducing the IC50 of the chemotherapeutic agent by at least 5-fold. In certain embodiments, the antibodies of the present disclosure may reduce the IC50 of the chemotherapeutic agent by 5-fold or more, e.g., 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, e.g., 5-10-fold.

[0061] In certain embodiments, one or more of the anti-ABCC1 antibodies disclosed herein bind to both human and cynomolgus monkey ABCC1, a property that can be utilized in determining the safety of the antibody in animal models.

[0062] In certain embodiments, the anti-ABCC1 antibodies disclosed herein are specific for ABCC1 and do not exhibit significant binding to other antigens.

[0063] In some embodiments, one or more of the subject antibodies may interfere with the function of cellular ABCC1 protein when bound to cells expressing ABCC1. Thus, one or more antibodies of the present disclosure may inhibit efflux by ABCC1 protein, including when efflux is reduced by 5% or more, including, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to efflux by ABCC1 in the absence of the subject antibody. In some embodiments, the subject antibodies, when bound to cells expressing ABCC1, may otherwise interfere with the action of ABCC1 by other mechanisms, such as leaking ABCC1, which may increase chemotherapeutic agent uptake and / or reduce cell viability.

[0064] In certain embodiments, anti-ABCC1 antibodies are provided that compete for binding to ABCC1 with an antibody comprising the heavy chain complementarity determining regions (HCDRs) and light chain CDRs (LCDRs) of the variable heavy chain (VH) and variable light chain (VL) region pairs, respectively, of an antibody listed in Table 2. For example, in one embodiment, an anti-ABCC1 antibody of the present disclosure competes for binding to ABCC1 with the C1.309 antibody listed in Table 2. In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0065] In certain embodiments, an anti-ABCC1 antibody comprises HCDR1, HCDR2, and HCDR3 of the VH region of an antibody listed in Table 2. In certain embodiments, HCDR1, HCDR2, and HCDR3 are defined according to the Kabat nomenclature. For example, in one embodiment, an anti-ABCC1 antibody of the present disclosure that competes for binding to ABCC1 with the C1.309 antibody listed in Table 2 comprises HCDR1, HCDR2, and HCDR3 of the VH region of the C1.309 antibody.

[0066] Any suitable approach can be used to determine whether a first antibody competes with a second antibody for binding to ABCC1. Whether a first antibody "competes with" a second antibody for binding to an antigen can be readily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, through antibody competition assays. For example, a sample of a first antibody can be bound to a solid support. A sample of a second antibody suspected of being able to compete with the first antibody is then added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies bind to separate and distinct sites on the antigen, the labeled antibody will bind to the same level regardless of whether the suspected competing antibody is present. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete, and the amount of labeled antibody bound to the antigen will decrease. If an excess of unlabeled antibody is present, very little, if any, labeled antibody will bind.

[0067] For purposes of this disclosure, a competing antibody is one that reduces the binding of an antibody to an antigen by about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more. Details of the procedures for conducting such competitive assays are well known in the art and can be found, for example, in Harlow and Lane, *Antibodies*, *A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, pp. 567-569, 1988, ISBN 0-87969-314-2. Such assays can be performed quantitatively by using purified antibodies. A standard curve can be established by titrating one antibody against itself; i.e., the same antibody is used for both the label and the competitor. The ability of an unlabeled competing antibody to inhibit the binding of a labeled antibody to an antigen can be titrated. The results can be plotted and the concentrations required to achieve the desired degree of binding inhibition can be compared.

[0068] In certain embodiments, an antibody that specifically binds to ABCC1 comprises (i) HCDRs 1-3 and light chain CDRs (LCDRs 1-3) of a pair of variable heavy (VH) and variable light (VL) chain regions of an antibody listed in Table 2, (ii) HCDRs 1-3 of the VH region of an antibody listed in Table 2, (iii) LCDRs 1-3 of the VH region of an antibody listed in Table 2, or (iv) HCDRs 1-3 of the VH region of a first antibody listed in Table 2 and LCDRs 1-3 of the VL region of a second antibody listed in Table 2. HCDRs and LCDRs may be defined based on the Kabat nomenclature.

[0069] In certain embodiments, antibodies of the present disclosure that specifically bind to human ABCC1 comprise the HCDR1, HCDR2, and HCDR3 sequences, and the LCDR1, LCDR2, and LCDR3 sequences of the antibodies listed in Table 2. In addition to binding to human ABCC1, one or more of the antibodies provided herein may bind to ABCC1 from other mammalian species, such as mouse, monkey, or chimpanzee. The antibodies may be raised in mouse or rat. Table 2 lists the animals from which the antibodies were generated. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19] [Table 2-20] [Table 2-21]

[0070] The anti-ABCC1 antibodies listed in Table 2 may also be referred to as anti-KPC1 antibodies or anti-C1 antibodies and may be referred to by the antibody numbers listed in Table 2.

[0071] In some embodiments, the antibody comprises a VL region and a VH region that are present in separate polypeptides, while in other embodiments, the VL region and the VH region are contained in a single polypeptide.

[0072] The antibody of the present disclosure may be selected from the group consisting of an Ig monomer, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an scFv, a scAb, a dAb, and an Fv.

[0073] In some embodiments, the subject antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized, or in vitro-generated antibody. As used herein, the term "recombinant" or "modified" antibody is intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell, (ii) antibodies isolated from a recombinant combinatorial antibody library, (iii) antibodies isolated from an animal (e.g., a mouse) transgenic with human immunoglobulin genes, or (iv) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies include humanized, CDR-grafted, chimeric, deimmunized, and in vivo-generated antibodies, and may optionally comprise constant regions derived from human germline immunoglobulin sequences.

[0074] As described above, a subject anti-ABCC1 antibody specifically binds to one or more epitopes of ABCC1. Thus, the epitopes are ABCC1 epitopes. The size of the ABCC1 epitopes bound by an anti-ABCC1 antibody can vary, including when the ABCC1 epitope is formed by a polypeptide having a continuous stretch of ABCC1 sequence that can range from 3 aa or less to 12 aa or more, including, but not limited to, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 4-10 aa, 5 aa-10 aa, 6 aa-10 aa, 4 aa-8 aa, 5 aa-8 aa, 6 aa-8 aa, etc.

[0075]

[0076] A subject anti-ABCC1 antibody exhibits high affinity binding to ABCC1. For example, a subject anti-ABCC1 antibody binds to human ABCC1 with an affinity of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M or 10 -12 The subject anti-ABCC1 antibodies bind to epitopes present on ABCC1 with an affinity of approximately 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, or about 10 -11 M~about 10 -12 M or 10 -12 It binds with an affinity greater than M.

[0077] A subject anti-ABCC1 antibody exhibits substantially no binding to any epitope formed by amino acids in other related but divergent proteins, such as related but divergent sequence EPs. Any binding of a subject anti-ABCC1 antibody to an epitope formed by amino acids in a related but divergent sequence protein is generally nonspecific binding with a substantially lower affinity than the specific binding of the anti-ABCC1 antibody to the epitope in ABCC1. A substantially lower affinity is generally at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower affinity.

[0078] A subject anti-ABCC1 antibody can reduce transport of a molecule through an ABCC1 transporter, e.g., human ABCC1. For example, a subject anti-ABCC1 antibody can reduce transport by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the extent of transport in the absence of the anti-ABCC1 antibody.

[0079] In some embodiments, the subject antibodies comprise FR regions that are mammalian sequences, including, for example, rodent, non-human primate, and human sequences (eg, encoded by respective heavy chain FR coding sequences).

[0080] The subject antibodies can comprise a heavy chain variable (VH) region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, including 100%, identical to the sequence for the VH region of a VH-VL pair of an antibody described in Table 2. The subject antibodies can comprise a light chain variable (VL) region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, including 100%, identical to the sequence for the VL region of a VH-VL region pair of an antibody described in Table 2.

[0081] In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of one of the C1.844, C1.851, C1.831, or C1.861 antibodies listed in Table 2.

[0082] In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of one of the C1.773 antibody, C1.773a antibody, C1.777a antibody, C1.784a antibody, C1.786a antibody, C1.787a antibody, C1.827 antibody, C1.830B antibody, C1.831 antibody, C1.835 antibody, C1.841 antibody, C1.844 antibody, C1.845 antibody, C1.847 antibody, C1.851 antibody, C1.855 antibody, C1.861 antibody, C1.863 antibody, C1.876 antibody, C1.877 antibody, or C1.879A antibody listed in Table 2.

[0083] In certain embodiments, an antibody of the present disclosure may reduce the IC50 of a chemotherapeutic agent (e.g., vincristine) by 5-fold or more, e.g., 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, or 20-fold or more, e.g., 5 to 50-fold. In one aspect, the antibody molecule may reduce the IC50 of a chemotherapeutic agent (e.g., vincristine) by 5-fold or more and may comprise HCDRs 1-3 and / or LCDRs 1-3 of the C1.851 antibody, C1.841 antibody, C1.861 antibody, C1.831 antibody, C1.786a antibody, C1.787a antibody, or C1.777 antibody, as listed in Table 2.

[0084] The regions and / or chains of the subject antibodies may or may not be joined by one or more linker regions. If present, the linker region can be from about 5 amino acids to about 50 amino acids in length, e.g., from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa in length.

[0085] Linkers suitable for use with the subject antibodies include "flexible linkers." When present, the linker molecule is generally long enough to allow some degree of flexible movement between the linked regions. Linker molecules are generally about 6-50 atoms in length. Linker molecules can be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules capable of binding to polypeptides can be used in view of the present disclosure.

[0086] Suitable linkers can be easily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0087] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , G.S.G.S.G.S. n (SEQ ID NO: 160), and GGGS n(SEQ ID NO: 161), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers are of particular interest because glycine has significantly more access to pi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 162), GGSGG (SEQ ID NO: 163), GSGSG (SEQ ID NO: 164), GSGGG (SEQ ID NO: 165), GGGSG (SEQ ID NO: 166), GSSSG (SEQ ID NO: 167), and the like. Those skilled in the art will recognize that the design of peptides conjugated to any of the above elements can include linkers that are wholly or partially flexible, whereby the linker can include a flexible linker as well as one or more moieties that impart a less flexible structure.

[0088] In other cases, the flexibility of the hinge region of the antibodies of the present disclosure can be reduced by mutating amino acid C220 to serine or any other natural amino acid, by removing C220, by removing the entire hinge, or by replacing the IgG1 hinge with an IgG3 hinge, forming an antibody in which the light chains are connected via their C-terminal cysteines, similar to the situation found in the human isotype IgA2m. This reduces the flexibility of the Fab to the Fc, resulting in a reduced cross-linking ability. Another strategy to reduce the flexibility of the IgG1 molecule is to replace the IgG1 hinge with an IgG2 hinge or an IgG2-like hinge. Alternatively, a variant of the IgG1 hinge similar to the IgG2 hinge can be introduced. This mutant (TH7Δ6-9) contains the mutation T223C and two deletions (K222 and T225) to generate a shorter hinge with an additional cysteine.

[0089] Substitution of mouse CDRs into a human variable domain framework can result in the retention of their correct spatial orientation; for example, the human variable domain framework adopts the same or a similar conformation as the mouse variable domain framework from which the CDRs were derived. This can be achieved by obtaining the human variable domain from a human antibody whose framework sequences show high sequence identity with the mouse variable framework domain from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be those of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

[0090] Now that the complementarity-determining regions of the murine donor immunoglobulin and the appropriate human acceptor immunoglobulin have been identified, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. Generally, substitution of murine for human amino acid residues should be minimized because the introduction of murine residues increases the risk of the antibody eliciting a human-anti-murine antibody (HAMA) response in humans. Art-recognized methods for determining immune responses can be performed to monitor HAMA responses in specific patients or during clinical trials. Patients receiving humanized antibodies can undergo immunogenicity assessments at the start of and throughout administration of the therapy. HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from patients using methods known to those skilled in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HAMA response in human subjects.

[0091] Specific amino acids from the human variable region framework residues are selected for substitution based on their potential effect on CDR conformation and / or antigen binding. The non-native juxtaposition of the murine CDR regions with the human variable framework regions can result in conformational constraints, resulting in reduced binding affinity unless corrected by substitution of specific amino acid residues.

[0092] The selection of amino acid residues for substitution can be determined, in part, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are generated starting from a solved structure of an immunoglobulin chain or its domain. The chain to be modeled is compared for amino acid sequence similarity with the chain or domain of the solved three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for building the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, preferably those sharing at least 60%, 70%, 80%, 90%, or more sequence identity. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain to be modeled and those in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0093] In some embodiments, the subject antibodies comprise scFv multimers. For example, in some embodiments, the subject antibodies are scFv dimers (e.g., comprising two tandem scFvs (scFv2)), scFv trimers (e.g., comprising three tandem scFvs (scFv3)), scFv tetramers (e.g., comprising four tandem scFvs (scFv4)), or multimers of more than four scFvs (e.g., in tandem). The scFv monomers can be linked in tandem via linkers of about 2 amino acids to about 15 amino acids in length, e.g., linkers of 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, or 15 aa in length. Suitable linkers include, for example, (Gly) x(SEQ ID NO: 168), where x is an integer between 2 and 15. Other suitable linkers are discussed above. In some embodiments, each of the scFv monomers in the subject scFv multimer is humanized as described above. In certain embodiments, the bispecific antibody can be in any molecular format known in the literature. For example, the bispecific antibody of the present disclosure can have the molecular format described in Spiess C. et al., Mol Immunol. 2015 Oct;67(2 Pt A):95-106.

[0094] In some embodiments, the subject antibodies comprise an immunoglobulin constant region (e.g., Fc region). The Fc region, if present, can be a human Fc region. If a constant region is present, the antibody can comprise both light and heavy chain constant regions. A suitable heavy chain constant region includes CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, and antibodies having any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is human isotype IgG1 Fc. The light chain constant region can be lambda or kappa. Antibodies of the invention (e.g., humanized antibodies of the invention) can comprise sequences from more than one class or isotype. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which the heavy and light chain variable domains are linked via a spacer.

[0095] In some embodiments, the subject antibodies comprise a free thiol (—SH) group at the carboxyl terminus, which can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a scaffold, a carrier, etc.

[0096] The antibody of interest can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the antibody of interest, a synthetic polymer, a carbohydrate, a toxin, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks polypeptides through their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and imidoesters crosslink amine-containing polypeptides. At mildly alkaline pH, imidoesters react only with primary amines to form imidoamides, leaving the overall charge of the crosslinked polypeptides unaffected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimidohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-bis[3-(2-pyridyldithio)propionamido]butane (DPDPB).

[0097] bispecific antibody In certain embodiments, the antibodies provided herein may be bispecific antibodies comprising a VH region and a VL region of an anti-ABCC1 antibody as described in the preceding section, and further comprising a second VH region comprising HCDRs 1-3 of an antibody that binds a tumor-associated antigen (TAA).

[0098] The antibody can comprise a second VL region, wherein the second VH region and the second VL region bind to a TAA. In certain embodiments, the second VH region and the second VL region can be present in a single polypeptide. In certain embodiments, the second VH region and the second VL region are present in an scFv.

[0099] In certain embodiments, the bispecific antibody comprises a common light chain, which comprises the VL region of an anti-ABCC1 antibody as described in the preceding section.

[0100] TAA can be any antigen known to be overexpressed in cancer cells.For example, TAA is an antigen that is not expressed at a detectable level in normal cells but is expressed in cancer cells, and normal cells and cancer cells are the same cell type, for example, epithelial cells.For example, TAA can be neoantigen, which is a class of tumor antigens that arise from tumor-specific mutations that change the amino acid sequence of encoded protein compared to the amino acid sequence of unmutated protein.In another embodiment, TAA is an antigen that is expressed in normal cells but is expressed at a higher level in cancer cells.

[0101] In certain embodiments, the TAA can be PD-L1. PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). In certain embodiments, the antibody that binds to PD-L1 can be atezolizumab.

[0102] In certain embodiments, a bispecific antibody may comprise a VH region comprising HCDRs 1-3 and a VL region comprising LCDRs 1-3, respectively, of the VH and VL regions of the C1.844 or C1.851 antibody as listed in Table 2, and a second VH region and a second VL region comprising HCDRs 1-3 and LCDRs 1-3, respectively, of atezolizumab. In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0103] In certain embodiments, the bispecific antibody comprises an scFv comprising the VH and VL regions of the C1.844 or C1.851 antibody listed in Table 2 and HCDRs 1-3 and LCDRs 1-3 of atezolizumab, where HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0104] In certain embodiments, a bispecific antibody may comprise a VH region comprising HCDRs 1-3 and a VL region comprising LCDRs 1-3, respectively, of the VH and VL regions of the C1.844hu21 or C1.851hu12 antibody as listed in Table 2, and a second VH region and a second VL region comprising HCDRs 1-3 and LCDRs 1-3, respectively, of atezolizumab. In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0105] In certain embodiments, the bispecific antibody comprises the VH and VL regions of the C1.844hu21 or C1.851hu12 antibody listed in Table 2 and an scFv comprising HCDRs 1-3 and LCDRs 1-3 of atezolizumab, where HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0106] In certain embodiments, HCDRs 1-3 present in the second VH region or scFv region of the bispecific antibody are HCDRs 1-3 of atezolizumab, where HCDR1 comprises the sequence DSWIH (SEQ ID NO: 25), HCDR2 comprises the sequence WISPYGGSTYYADSVKG (SEQ ID NO: 169), and HCDR3 comprises the sequence RHWPGGFDY (SEQ ID NO: 170). In certain embodiments, a bispecific antibody that binds to ABCC1 and PD-L1 may further comprise a second VL region comprising LCDRs 1-3 of atezolizumab, where LCDR1 comprises the sequence RASQDVSTAVA (SEQ ID NO: 171), LCDR2 comprises the sequence SASFLYS (SEQ ID NO: 172), and LCDR3 comprises the sequence QQYLYHPAT (SEQ ID NO: 173). In certain embodiments, LCDRs 1-3 present in the scFv region comprise LCDRs 1-3 of atezolizumab, as defined according to the Kabat nomenclature.

[0107] In certain embodiments, a bispecific antibody that binds to ABCC1 and PD-L1 has the amino acid sequence of the VH region of atezolizumab as set forth below: and a second VH region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, or 100% identical to EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 174).

[0108] In certain embodiments, a bispecific antibody that binds to ABCC1 and PD-L1 has the amino acid sequence of the VL region of atezolizumab as set forth below: and a second VL region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, or 100% identical to Diqmtqspsslsasvgdrvtitcrasqdvstavawyqqkpgkapklliysasflysgvpsrfsgsgsgtdftltisslqpedfatyycqqylyhpatfgqgtkveik (SEQ ID NO: 175).

[0109] In certain embodiments, the bispecific antibody that binds to ABCC1 and PD-L1 binds to PD-L1 and has the amino acid sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK (SEQ ID NO: 176).

[0110] The italicized sequence is the linker sequence between the VH and VL regions. Any other linker sequence can be used to connect the VH and VL regions.

[0111] In certain embodiments, the TAA is ErbB2 (HER2). ErbB2 is also known as receptor tyrosine kinase 2 or HER2. In certain embodiments, the antibody that binds to ErbB2 is trastuzumab.

[0112] In certain embodiments, the bispecific antibody comprises a VH region comprising HCDRs 1-3 and a VL region comprising LCDRs 1-3 of the VH and VL regions, respectively, of the C1.844, C1.831, or C1.851 antibodies listed in Table 2, and a second VH region and a second VL region comprising HCDRs 1-3 and LCDRs 1-3, respectively, of trastuzumab. In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0113] In a specific embodiment, the bispecific antibody comprises a VH region comprising HCDRs 1 to 3 and a VL region comprising LCDRs 1 to 3 of the VH region and VL region of the C1.844 antibody, the C1.831 antibody, or the C1.851 antibody, respectively, listed in Table 2, and an scFv comprising HCDRs 1 to 3 and LCDRs 1 to 3 of trastuzumab.

[0114] In certain embodiments, the bispecific antibody comprises the VH and VL regions of the C1.844hu21 antibody, the C1.831hu11 antibody, or the C1.851hu12 antibody, and an scFv comprising HCDRs 1-3 and LCDRs 1-3 of trastuzumab, where HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0115] In certain embodiments, HCDRs 1-3 present in the second VH region or scFv region of the bispecific antibody are HCDRs 1-3 of trastuzumab, where HCDR1 comprises the sequence DTYIH (SEQ ID NO: 177), HCDR2 comprises the sequence RIYPTNGYTRYADSVKG (SEQ ID NO: 178), and HCDR3 comprises the sequence WGGDGFYAMDY (SEQ ID NO: 179). In certain embodiments, a bispecific antibody that binds to ABCC1 and HER2 may further comprise a second VL region comprising LCDRs 1-3 of trastuzumab, where LCDR1 comprises the sequence RASQDVNTAVA (SEQ ID NO: 180), LCDR2 comprises the sequence SASFLYS (SEQ ID NO: 172), and LCDR3 comprises the sequence QQHYTTPPT (SEQ ID NO: 181). In certain embodiments, LCDRs 1-3 present in the scFv region comprise LCDRs 1-3 of trastuzumab, as defined according to the Kabat nomenclature.

[0116] In a particular embodiment, a bispecific antibody that binds to ABCC1 and HER2 has the amino acid sequence of the VH region of trastuzumab as set forth below: and a second VH region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, or 100% identical to Evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsvkgrftisadtskntaylqmnslraedtavyycsrwggdgfyamdywgqgtlvtvss (SEQ ID NO: 182).

[0117] In certain embodiments, a bispecific antibody that binds to ABCC1 and HER2 has the amino acid sequence of the VL region of trastuzumab as set forth below: and a second VL region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, or 100% identical to Diqmtqspsslsasvgdrvtitcrasqdvntavawyqqkpgkapklliysasflysgvpsrfsgsrsgtdftltisslqpedfatyycqqhyttpptfgqgtkveik (SEQ ID NO: 183).

[0118] In certain embodiments, the bispecific antibody that binds to ABCC1 and HER2 binds to HER2 and has the amino acid sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGCGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKCPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 184).

[0119] The italicized sequence is the linker sequence between the VH and VL regions. Any other linker sequence can be used to connect the VH and VL regions.

[0120] In certain embodiments, the heavy chain of the bispecific antibody may comprise a VH region and a heavy chain constant region as provided herein. In certain embodiments, the light chain of the bispecific antibody may comprise a VL region and a light chain constant region as provided herein. In certain embodiments, the scFv may be conjugated to the heavy chain constant region.

[0121] The heavy and light chain constant regions can be those of a human IgG antibody, e.g., a human IgG1 antibody. The constant regions can have wild-type or modified sequences. In some embodiments, the bispecific antibody can comprise a modified heavy chain comprising a modified Fc domain comprising a modified CH2 and / or CH3 domain. In some cases, the modified Fc domain can utilize electrostatic steering effects, including, but not limited to, by using the procedures described in Gunasekeran et al. (2010) Journal of Biological Chemistry 285, 19637-19646 (the disclosure of which is incorporated herein by reference in its entirety). In some cases, the bispecific antibody is constructed by charge-pair substitutions in the CH3 domain, including, but not limited to, when one heavy chain is modified to include K392D and K409D substitutions and the other heavy chain is modified to include E356K and D399K substitutions. Charge-pair substituted chains may preferentially form heterodimers with each other. The numbering of amino acid substitutions follows the EU numbering system of HC.

[0122] In some cases, the antibodies of the present disclosure comprise charge pair substitutions. In some cases, the antibodies of the present disclosure do not comprise charge pair substitutions. In some cases, alternative means of promoting preferential heterodimer formation of the desired chains can be used.

[0123] In some cases, the modified heavy chain may include a knob-into-hole modification. Knob-into-hole amino acid modification is a rational design strategy in antibody engineering and is used for heavy chain heterodimerization in the production of multispecific antibodies, including bispecific IgG antibodies. For example, when incorporating the knob-into-hole strategy into a bispecific antibody made from two monoclonal antibodies of different specificities, amino acid changes are engineered to create a "knob" in the CH3 of the heavy chain of monoclonal antibody 1 (mAb1) and a "hole" in the CH3 of the heavy chain of monoclonal antibody 2 (mAb2). The knob may be represented by a large amino acid, such as tyrosine (Y), while the hole may be represented by a small amino acid, such as threonine (T). For example, a knob-into-hole modification may create a T22Y substitution in the first CH3 domain and a Y86T substitution in the partner CH3 domain. Examples of knob-into-hole modifications are described in Carter, J. Immunol. Methods, 248(1-2):7-15 (2001); Ridgway, J. B. et al. Protein Eng. 9(7):617-2 (1996); and Merchant, A. M. et al. Nat. Biotechnol. 16(7):677-81 (1998), the disclosures of which are incorporated herein in their entireties. In antibodies generated from paired knob-into-hole modified domains, bispecific heterodimers generally represent the majority.

[0124] In certain embodiments, the bispecific antibodies provided herein bind to cancer cells that express both ABCC1 and a TAA, while exhibiting reduced binding to non-cancer cells that express ABCC1 and / or a TAA. In other words, the bispecific antibodies provided herein bind with low affinity to (1) cells that express a TAA, where ABCC1 expression is low or absent, and (2) cells that express ABCC1, where TAA expression is low or absent, and bind with high affinity to cancer cells that express at least one or both of ABCC1 and a TAA at relatively high levels, i.e., at levels higher than normal cells.

[0125] Compositions and Formulations The present disclosure provides compositions comprising a subject antibody. The subject antibody compositions can include, in addition to the subject antibody, one or more of the following: a salt, e.g., NaCl, MgCl, KCl, MgSO, etc.; a buffering agent, e.g., Tris buffer, histidine buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20; a protease inhibitor; glycerol, etc.

[0126] The compositions of the present disclosure also include pharmaceutical compositions comprising the antibodies described herein. Generally, the formulation contains an effective amount of the subject antibody. An "effective amount" refers to a dosage sufficient to produce a desired result, such as a reduction in cancer in a subject, a reduction in the growth rate of cancer in a subject, or an improvement in the symptoms of cancer. Generally, the desired result is at least a reduction in the symptoms of cancer, a reduction in the growth of cancer, or a reduction in the size of cancer compared to a control. The subject antibody can be delivered or formulated in a way that bypasses the blood-brain barrier.

[0127] In some cases, the antibody may include a delivery enhancer, where such an enhancer may facilitate crossing of the blood-brain barrier, for example, by increasing permeability to allow for efficient transdermal delivery.

[0128] In some cases, the antibody of the present disclosure does not need to be administered in a formulation with a delivery enhancer. In some cases, the antibody of the present disclosure may itself enhance permeability across the blood-brain barrier. In some cases, the antibody of the present disclosure may be used as a delivery enhancer to promote crossing of the blood-brain barrier by an anti-tumor agent, such as an immunotherapeutic agent or a chemotherapeutic agent. In some cases, the antibody of the present disclosure may be used as a delivery enhancer to promote crossing of the blood-brain barrier, blood-cerebrospinal fluid (CSF) barrier, blood-testis barrier, or blood-placental barrier by an active agent, such as another antibody or a chemotherapeutic agent.

[0129] In the subject method, the subject antibody can be administered to a host using any convenient means that can produce the desired therapeutic or diagnostic effect. Thus, the agent can be incorporated into various formulations for therapeutic administration. More specifically, the subject antibody can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0130] In pharmaceutical dosage forms, the subject antibodies can be administered in conjunction with pharmaceutically acceptable excipients, or they can be used alone or in appropriate association and combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0131] The subject antibodies can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, together with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed.

[0132] Pharmaceutical compositions containing the subject antibodies are prepared by mixing the antibody having the desired purity with, optionally, physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, and the like. amino acids such as thiamin, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0133] The pharmaceutical compositions may be in liquid form, lyophilized form, or in liquid form reconstituted from lyophilized form, with lyophilized preparations being reconstituted with a sterile solution prior to administration.

[0134] Exemplary antibody concentrations in a subject pharmaceutical composition can range from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0135] Aqueous antibody formulations can be prepared in pH buffer solutions at pHs ranging from about 4.0 to about 7.5, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for pHs within this range include phosphate, histidine, citrate, succinate, acetate, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0136] In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as physiological salt solution or serum. Isotonic agents may be used in amounts of about 5 mM to about 350 mM, e.g., 100 mM to 350 nM.

[0137] Surfactants may also be added to antibody formulations to reduce aggregation of the formulated antibody and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Exemplary concentrations of surfactants may range from about 0.001% to about 1% w / v.

[0138] Cryoprotectants may also be added to protect unstable active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Cryoprotectants may be included in amounts of about 10 mM to 500 nM.

[0139] In some embodiments, a subject formulation comprises a subject antibody and one or more of the above-identified agents (e.g., surfactant, buffer, stabilizer, isotonicity agent), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0140] For example, a subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration, and can contain from about 1 mg / mL to about 200 mg / mL of a subject antibody, from about 0.001% to about 1% of at least one surfactant, from about 1 mM to about 100 mM of a buffering agent, optionally from about 10 mM to about 500 mM of a stabilizer, and from about 5 mM to about 305 mM of an isotonic agent, and has a pH of from about 4.0 to about 7.0.

[0141] The subject antibodies can be utilized in aerosol formulations to be administered via inhalation. The subject antibodies can be formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0142] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound of the invention calculated to be sufficient to produce a desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the subject antibody may depend on the particular antibody employed and the effect to be achieved, as well as the pharmacodynamics associated with each antibody in the host.

[0143] The subject antibodies can be administered as injectable formulations. Typically, injectable compositions are prepared as liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. Preparations can also be emulsified, or the antibodies can be encapsulated in liposome vehicles.

[0144] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods for preparing such dosage forms are known, or will be apparent, to those skilled in the art.

[0145] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0146] In some embodiments, the subject antibodies are formulated in a controlled release formulation. Sustained release preparations can be prepared using methods well known in the art.

[0147] Dosage The appropriate dosage can be determined by the attending physician or other qualified medical professional based on various clinical factors. As is well known in the medical field, the dosage for any single patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, the patient's sex, duration, and route of administration, general health, and other drugs being administered concomitantly. The subject antibodies may be administered in amounts of 1 ng / kg to 20 mg / kg body weight, e.g., 0.1 mg / kg to 10 mg / kg body weight, e.g., 0.5 mg / kg to 5 mg / kg body weight per dose, although doses below or above this exemplary range are contemplated, particularly considering the aforementioned factors. If the regimen is a continuous infusion, it can also range from 1 μg to 10 mg per kilogram of body weight per minute.

[0148] Those of skill in the art will readily appreciate that dosage levels can vary as a function of the particular antibody, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0149] Route of administration The subject antibodies are administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local routes of administration.

[0150] Conventional pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined or adjusted as needed depending on the antibody and / or the desired effect. A subject antibody composition may be administered in a single dose or multiple doses. In some embodiments, a subject antibody composition is administered orally. In some embodiments, a subject antibody composition is administered via the inhalation route. In some embodiments, a subject antibody composition is administered intranasally. In some embodiments, a subject antibody composition is administered topically. In some embodiments, a subject antibody composition is administered intracranially. In some embodiments, a subject antibody composition is administered intravenously.

[0151] The agents can be administered to a host using any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. Generally, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0152] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can be carried out to effect systemic or local delivery of the subject antibody. When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of the pharmaceutical preparation.

[0153] A subject antibody can also be delivered to a subject by enteral administration, which routes of enteral administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

[0154] Treatment refers to at least an amelioration of symptoms associated with a pathological condition afflicting the host, where amelioration is used broadly to refer to at least a decrease in the magnitude of parameters, e.g., symptoms, associated with the pathological condition being treated, such as cancer and / or cancer growth and associated pain, etc. Thus, treatment also includes situations where the pathological condition, or at least the symptoms associated therewith, are completely inhibited, e.g., prevented from occurring, or halted, e.g., terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms characterized by the pathological condition.

[0155] A variety of subjects (the term "subject" is used interchangeably herein with the terms "individual" and "patient") can be treated according to the methods of the present disclosure. Generally, such subjects are "mammals" or "mammals," which terms are used broadly to describe organisms within the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodents (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host is a human.

[0156] Kits are provided having unit doses of the subject antibodies, e.g., oral or injectable doses. In some embodiments, in addition to the container containing the unit dose is an information package insert describing the use of the antibody in treating the pathological condition of interest and the associated benefits.

[0157] nucleic acid The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody of interest, which can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow for expression of the nucleotide sequence in an intended target cell (e.g., a cell that has been genetically engineered to synthesize and / or secrete the encoded antibody).

[0158] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, the light and / or heavy chain immunoglobulin gene promoter and enhancer elements, the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoters, promoters found in the long terminal repeats from retroviruses, the mouse metallothionein-I promoter, and various art-known tissue-specific promoters.

[0159] The nucleotide sequence encoding the antibody of interest can be present in an expression vector and / or a cloning vector. If the antibody of interest comprises two or more separate polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned into the same or separate vectors. Separate polypeptides can be expressed from a single nucleic acid or a single vector using a variety of strategies, including separate promoters, one or more internal ribosome entry sites (IRES), one or more self-cleaving sequences (e.g., 2A cleavage sequences such as P2A, T2A, E2A, and F2A), or combinations thereof. Expression vectors can include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector.

[0160] Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0161] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus), retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like.

[0162] For example, nucleic acid as described herein can be introduced into cells in some cases, for example, by contacting cells with nucleic acid.Cells with introduced nucleic acid are generally referred to herein as genetically modified cells.Various nucleic acid delivery methods can be used, including but not limited to, naked nucleic acid delivery, viral delivery, chemical transfection, gene gun, etc.

[0163] cell The present disclosure provides isolated genetically modified cells (e.g., in vitro cells, ex vivo cells, cultured cells, etc.) that have been genetically modified with a nucleic acid of interest. In some embodiments, the isolated genetically modified cells of interest are capable of producing an antibody of interest. In some cases, the genetically modified cells are capable of delivering the antibody, for example, to a subject in need thereof.

[0164] Suitable cells include eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as bacterial cells. Introduction of a nucleic acid of interest into a host cell can be affected, for example, by calcium phosphate precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0165] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells, CHO cells, 293 cells, 3T3 cells, Vero cells, Huh-7 cells, BHK cells, PC12 cells, COS cells, COS-7 cells, RAT1 cells, mouse L cells, human embryonic kidney (HEK) cells, HLHepG2 cells, etc.

[0166] In some cases, useful mammalian cells can include cells derived from mammalian tissues or organs. In some cases, the cells used are kidney cells, including, for example, kidney cells from established kidney cell lines such as HEK293T cells.

[0167] In some cases, the cells of the present disclosure may be immune cells. As used herein, the term "immune cells" generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.

[0168] In some cases, useful cells expressing antibodies, such as the multispecific antibodies of the present disclosure, may include producer T cells. Producer T cells engineered to contain nucleic acid sequences encoding the antibodies of the present disclosure may, in some cases, be used to deliver the antibodies to a subject in need thereof.

[0169] In some cases, the immune cells of the present disclosure include immune effector cells comprising a chimeric antigen receptor (CAR) comprising an ABCC1-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the ABCC1-binding domain comprises the heavy chain complementarity-determining region (HCDR) and light chain CDR (LCDR) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. In one embodiment, the intracellular signaling domain can comprise one or more functional signaling domains derived from at least one costimulatory molecule, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. The intracellular signaling domain can comprise a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.

[0170] The immune effector cells can be T cells. The immune effector cells can be autologous cells.

[0171] method As summarized above, the methods of the present disclosure include methods of contacting a cell with an antibody of the present disclosure, methods of treating a subject according to methods involving administering to the subject an antibody of the present disclosure, and methods of making the elements described in this application, e.g., antibodies, compositions and formulations, nucleic acids, expression vectors, cells, etc.

[0172] As summarized above, the methods of the present disclosure include contacting cancer cells with an antibody of the present disclosure to, for example, detect the presence of ABCC1 expression on the cancer cells, measure the level of ABCC1 expression on the cancer cells, or promote and / or enhance cancer cell killing. In some cases, cancer cell killing is mediated by an immune response or immune cells acting on cancer cells bound by the antibody. In some cases, cancer cell killing is mediated by inhibition of cancer cell cytotoxicity, e.g., as a result of antibody-mediated ABCC1 inhibition. In some cases, cancer cell killing is mediated by a combination of inhibition of cancer cell cytotoxicity and an immune-mediated response (e.g., via the Fc region of the antibody). Methods involving contacting cancer cells with an antibody of the present disclosure may or may not include contacting the cancer cells with an additional therapy or active agent, including, for example, chemotherapy, immunotherapy, radiation therapy, etc.

[0173] Treatment method The present disclosure provides methods of treating cancer, which generally involve administering to an individual in need thereof (e.g., an individual with cancer) an effective amount of an antibody as provided herein, alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy). Administration of the antibodies of the present disclosure can be by any convenient, suitable route of delivery.

[0174] Thus, administration includes, for example, but is not limited to, delivery of an antibody by injection, delivery of an antibody by infusion, delivery of a nucleic acid or expression vector encoding the antibody, delivery of an antibody by administering to a subject cells that express and secrete the antibody, delivery of immune effector cells (e.g., CAR-T cells) that express a chimeric antigen receptor (CAR) on the cell surface comprising an ABCC1-binding domain, a transmembrane domain, and an intracellular signaling domain, where the ABCC1-binding domain comprises, for example, the HCDR and LCDR of a pair of VH and VL regions of an antibody listed in Table 2. Administration of an agent, a nucleic acid encoding an agent, a cell expressing an agent, etc. can include contacting with the agent, contacting with a nucleic acid, contacting with a cell, etc.

[0175] In some embodiments, an effective amount of a subject antibody, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is an amount effective to reduce adverse symptoms of cancer by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the severity of the adverse symptoms in the absence of treatment with the antibody.

[0176] In some embodiments, an effective amount of a subject antibody, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is an amount effective to ameliorate cancer (i.e., slow the growth of cancer, stop the growth of cancer, reverse the growth of cancer, kill cancer cells (including tumor cells, etc.)) in the individual being treated. For example, an effective amount of a subject antibody can reduce the rate of cancer growth in an individual or reduce the size of a cancer in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more, compared to the absence of antibody treatment.

[0177] In some cases, a subject may be treated systemically, including using a subject antibody with or without one or more additional reagents. As used herein, "systemic treatment" refers to a treatment that does not only target a specific tumor (e.g., a primary tumor or a defined secondary tumor) or a specific cancer-containing tissue (e.g., the liver in the case of liver cancer, the blood in the case of blood cancer, etc.). Systemic treatment is generally directed to the subject's body as a whole and may include, for example, but is not limited to, systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof, etc.

[0178] In some cases, a subject may be treated locally, including using a subject antibody with or without one or more additional reagents. "Local treatment," as used herein, refers to treatment that is specifically directed to the location of the tumor (e.g., the primary tumor or a defined secondary tumor) or to the tissue containing the cancer (e.g., the liver in the case of liver cancer, the blood in the case of blood cancer, etc.). In some cases, a local treatment may also be administered in a manner that affects the environment surrounding the tumor, such as tissue surrounding the tumor, such as tissue immediately adjacent to the tumor. A local treatment generally does not affect or target tissues distant from the cancer site, including the tumor site, such as the primary tumor. Useful local treatments that can be administered in addition to or in combination with a subject antibody include, but are not limited to, surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, etc.

[0179] In some embodiments, a subject treatment method involves administering a subject antibody and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, chemotherapeutic agents, radiation therapy reagents, immunotherapy reagents, other antibody agents, etc. Additional therapies that may be administered to a subject before, during, or after administration of an antibody of the present disclosure to a subject will vary depending on numerous factors, including, for example, the type of cancer, the subject's medical history, general health, and / or any co-morbidities. Useful cancer therapies include, but are not limited to, for example, radiation therapy, chemotherapy, immunotherapy, etc.

[0180] Radiation therapy includes, but is not limited to, X-rays or gamma rays delivered from an externally applied source such as a beam or by implantation of small radioactive sources.

[0181] Suitable antibodies for use in cancer therapy include naked antibodies, such as trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), oregovomab (OvaRex™), lamotrigine (Lupin™), and ribozyme (Ribozyme™). These include, but are not limited to, mbrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), and conjugated antibodies such as gemtuzumab ozogamicin (Mylortarg™), brentuximab vedotin (Adcetris™), 90Y-labeled ibritumomab tiuxetan (Zevalin™), 131I-labeled tositumomab (Bexxar™), and the like.

[0182] Antibodies suitable for use in cancer therapy also include, but are not limited to, antibodies raised against tumor-associated antigens, such as CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucins, TAG-72, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Ley, VEGF, VEGFR, integrin alpha-V-beta-3, integrin alpha-5-beta-1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, and TRAILR1. , TRAILR2, RANKL, FAP, tenascin, programmed death-ligand 1 (PD-L1), androgen receptor (AR), Bruton's tyrosine kinase (BTK), BCR-Abl, c-kit, PIK3CA, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEKJ, MEK2, NRAS, RAC1, ESR1, CTLA-4, LAG-3, and TIM-3. These antibodies may be administered as combination therapy with the anti-ABCC1 antibodies provided herein.

[0183] Traditional cancer therapies also include targeted therapies against cancer, such as Ado-trastuzumab emtansine (Kadcyla) (approved for use in breast cancer) targeting HER2 (ERBB2 / neu), afatinib (Gilotrif) (approved for use in non-small cell lung cancer) targeting EGFR (HER1 / ERBB1), HER2 (ERBB2 / neu), aldesleukin (Proleukin) (approved for use in renal cell carcinoma, melanoma), and alectinib (Alecensa) targeting ALK. (approved for use in non-small cell lung cancer), alemtuzumab (Campath) targeting CD52 (approved for use in B-cell chronic lymphocytic leukemia), atezolizumab (Tecentriq) targeting PD-L1 (approved for use in urothelial carcinoma and non-small cell lung cancer), avelumab (Bavencio) targeting PD-L1 (approved for use in Merkel cell carcinoma), axitinib (Inlyta) targeting KIT, PDGFRβ, and VEGFR1 / 2 / 3 (approved for use in renal cell carcinoma), and BAF belimumab (Benlysta) targets F (approved for use in lupus erythematosus), belinostat (Beleodaq) targets HDAC (approved for use in peripheral T-cell lymphoma), bevacizumab (Avastin) targets VEGF ligands (approved for use in cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma, non-small cell lung cancer, ovarian cancer, peritoneal cancer, and renal cell cancer), and blinatumomab (Blincyto) targets CD19 / CD3 (approved for use in acute lymphoblastic leukemia (precursor B-cell)). ), bortezomib (Velcade) targeting the proteasome (approved for use in multiple myeloma and mantle cell lymphoma), bosutinib (Bosulif) targeting ABL (approved for use in chronic myeloid leukemia), brentuximab vedotin (Adcetris) targeting CD30 (approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma), brigatinib (Alunbrig) targeting ALK (approved for use in non-small cell lung cancer (ALK+)), FLT3, KIT, MET, RET,Cabozantinib (Cabometyx, Cometriq) targets VEGFR2 (approved for use in medullary thyroid cancer and renal cell carcinoma), carfilzomib (Kyprolis) targets the proteasome (approved for use in multiple myeloma), ceritinib (Zykadia) targets ALK (approved for use in non-small cell lung cancer), cetuximab (Erbitux) targets EGFR (HER1 / ERBB1) (approved for use in colorectal cancer and squamous cell carcinoma of the head and neck), and MEK targets cobimetinib (Cotellic) (approved for use in melanoma), which targets ALK, MET, and ROS1; crizotinib (Xalkori) (approved for use in non-small cell lung cancer), which targets BRAF; dabrafenib (Tafinlar) (approved for use in melanoma and non-small cell lung cancer), which targets CD38; daratumumab (Darzalex) (approved for use in multiple myeloma), which targets CD38; and dasatinib (Sprycel) (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia), which targets ABL. (approved for use in giant cell tumor of bone), denosumab (Xgeva) targeting RANKL (approved for use in giant cell tumor of bone), dinutuximab (Unituxin) targeting B4GALNT1 (GD2) (approved for use in pediatric neuroblastoma), durvalumab (Imfinzi) targeting PD-L1 (approved for use in urothelial carcinoma), elotuzumab (Empliciti) targeting SLAMF7 (CS1 / CD319 / CRACC) (approved for use in multiple myeloma), and enasideni targeting IDH2. Idhifa (approved for use in acute myeloid leukemia), erlotinib (Tarceva) which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer and pancreatic cancer), everolimus (Afinitor) which targets mTOR (approved for use in neuroendocrine tumors of pancreatic, gastrointestinal, or pulmonary origin, renal cell carcinoma, unresectable subependymal giant cell astrocytoma, and breast cancer), and gefitinib (Iressa) which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer).Ibritumomab tiuxetan (Zevalin) targets CD20 (approved for use in non-Hodgkin's lymphoma), ibrutinib (Imbruvica) targets BTK (approved for use in mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia), idelalisib (Zydelig) targets PI3Kδ (approved for use in chronic lymphocytic leukemia, follicular B-cell non-Hodgkin's lymphoma, and small lymphocytic lymphoma), and imatinib (Gleevec) targets KIT, PDGFR, and ABL. Approved for use in GI stromal tumors (KIT+), dermatofibrosarcoma protuberans, and multiple hematologic malignancies; ipilimumab (Yervoy) targeting CTLA-4 (approved for use in melanoma); ixazomib (Ninlaro) targeting the proteasome (approved for use in multiple myeloma); lapatinib (Tykerb) targeting HER2 (ERBB2 / neu) and EGFR (HER1 / ERBB1) (approved for use in breast cancer (HER2+)); and lenvatinib (Lenvima) targeting VEGFR2 (renal cell carcinoma). , approved for use in thyroid cancer), midostaurin (Rydapt) which targets FLT3 (approved for use in acute myeloid leukemia (FLT3+)), necitumumab (Portrazza) which targets EGFR (HER1 / ERBB1) (approved for use in squamous non-small cell lung cancer), neratinib (Nerlynx) which targets HER2 (ERBB2 / neu) (approved for use in breast cancer), nilotinib (Tasigna) which targets ABL (approved for use in chronic myeloid leukemia), and PARP Niraparib (Zejula) targets PD-1 (approved for use in ovarian, fallopian tube, and peritoneal cancer); nivolumab (Opdivo) targets PD-1 (approved for use in colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, melanoma, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma); obinutuzumab (Gazyva) targets CD20 (approved for use in chronic lymphocytic leukemia and follicular lymphoma); and ofatumumab (Arzerra, HuMax-CD20) targets CD20 (approved for use in chronic lymphocytic leukemia).Olaparib (Lynparza) targets PARP (approved for use in ovarian cancer), olaratumab (Lartruvo) targets PDGFRα (approved for use in soft tissue sarcoma), osimertinib (Tagrisso) targets EGFR (approved for use in non-small cell lung cancer), palbociclib (Ibrance) targets CDK4 and CDK6 (approved for use in breast cancer), and panitumumab (Vectibix) targets EGFR (HER1 / ERBB1) panobinostat (Farydak) which targets HDAC (approved for use in multiple myeloma); pazopanib (Votrient) which targets VEGFR, PDGFR, and KIT (approved for use in renal cell carcinoma); pembrolizumab (Keytruda) which targets PD-1 (approved for use in classical Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+), squamous cell carcinoma of the head and neck, and solid tumors (MSI-H)); and HER2 (ERBB2 / neu) Pertuzumab (Perjeta) targets breast cancer (approved for use in HER2+), ponatinib (Iclusig) targets ABL, FGFR1-3, FLT3, and VEGFR2 (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia), ramucirumab (Cyramza) targets VEGFR2 (approved for use in colorectal cancer, gastric cancer or esophagogastric junction (GEJ) adenocarcinoma, and non-small cell lung cancer), and KIT, PDGFRβ, RAF, RET, and VEGFR1 / 2 / 3. regorafenib (Stivarga) (approved for use in colorectal cancer, gastrointestinal stromal tumors, and hepatocellular carcinoma); ribociclib (Kisqali) (approved for use in breast cancer (HR+, HER2-)) targeting CDK4 and CDK6; rituximab (Rituxan, Mabthera) (approved for use in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis) targeting CD20; and rituximab / hyaluronidase human (Rituxan Hycela) (approved for use in chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma) targeting CD20.Romidepsin (Istodax) targets HDAC (approved for use in cutaneous T-cell lymphoma and peripheral T-cell lymphoma), rucaparib (Rubraca) targets PARP (approved for use in ovarian cancer), ruxolitinib (Jakafi) targets JAK1 / 2 (approved for use in myelofibrosis), siltuximab (Sylvant) targets IL-6 (approved for use in multicentric Castleman disease), and sipuleucel-T (Provenge) targets prostate cancer. approved for use in basal cell carcinoma), sonidegib (Odomzo) which targets smooth muscle (approved for use in basal cell carcinoma), sorafenib (Nexavar) which targets VEGFR, PDGFR, KIT, and RAF (approved for use in hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer), temsirolimus (Torisel) which targets mTOR (approved for use in renal cell carcinoma), tositumomab (Bexxar) which targets CD20 (approved for use in non-Hodgkin lymphoma), and MEK Trametinib (Mekinist) (approved for use in melanoma and non-small cell lung cancer), trastuzumab (Herceptin) (approved for use in breast cancer (HER2+) and gastric cancer (HER2+)) which targets HER2 (ERBB2 / neu), vandetanib (Caprelsa) (approved for use in medullary thyroid cancer) which targets EGFR (HER1 / ERBB1), RET, and VEGFR2, and vemurafenib (Zelboraf) (approved for use in melanoma) which targets BRAF. These antibodies include, but are not limited to, venetoclax (Venclexta) which targets BCL2 (approved for use in chronic lymphocytic leukemia), vismodegib (Erivedge) which targets PTCH and smooth muscle (approved for use in basal cell carcinoma), vorinostat (Zolinza) which targets HDAC (approved for use in cutaneous T-cell lymphoma), and Ziv-aflibercept (Zaltrap) which targets PIGF and VEGFA / B (approved for use in colorectal cancer). These antibodies may be administered as combination therapy with the anti-ABCC1 antibodies provided herein.

[0184] Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity, (2) inhibitors of serine / threonine kinase activity, (3) tumor-associated antigen antagonists such as antibodies that specifically bind to tumor antigens, (4) apoptosis receptor agonists, (5) interleukin-2, (6) interferon-α, (7) interferon-γ, (8) colony-stimulating factors, (9) inhibitors of angiogenesis, and (10) antagonists of tumor necrosis factor.

[0185] Chemotherapeutic or antitumor agents are non-peptide (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents (e.g., nitrosoureas), antimetabolites (e.g., methotrexate), antitumor antibiotics (e.g., anthracyclines), plant alkaloids (e.g., vinca alkaloids, taxanes, etc.), toposiomerase inhibitors, and steroid hormones.

[0186] Drugs that act to reduce cell proliferation are known in the art and are widely used. Such drugs include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkylsulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0187] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0188] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics, such as anthracyclines, daunorubicin hydrochloride, and the like. These include, but are not limited to, phenoxyzolinone cyclopeptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrroloindoleziones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like.

[0189] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0190] Microtubule-affecting agents with antiproliferative activity are also suitable for use, including, but not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterol, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones (including, but not limited to, etopirone A, epothilone B, discodermolide), estramustine, and nocodazole.

[0191] Hormonal modulating agents and steroids (including synthetic analogs) suitable for use include, but are not limited to, adrenocorticosteroids such as prednisone, dexamethasone, and the like; estrogens and pregestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; adrenocortical suppressants such as aminoglutethimide, 17α-ethinylestradiol, diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testolone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex. Estrogen stimulates proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0192] Other chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epidophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, and the like. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporin, leflunomide, mizoribine, azaspirane (SKF105685); Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline, and the like.

[0193] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be prepared by techniques known to those skilled in the art (WO 94 / 07882, WO 94 / 0788 1, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076, U.S. Pat. Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP 590,267 (see also), or can be obtained from a variety of commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis).

[0194] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., Taxotere™ docetaxel, as described above), and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, or protein-bound paclitaxel such as Abraxane®).

[0195] The term "taxane" also encompasses various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. 99 / 18113, the piperazino and other derivatives described in WO 99 / 14209, the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680, the 6-thio derivatives described in WO 98 / 28288, the sulfenamide derivatives described in U.S. Patent No. 5,821,263, and the taxol derivatives described in U.S. Patent No. 5,415,869. Prodrugs of paclitaxel are also included, including, but not limited to, those described in WO 98 / 58927, WO 98 / 13059, and U.S. Patent No. 5,824,701.

[0196] Useful immunotherapies include anti-PD-1 / PD-L1 immunotherapies and / or other immunotherapies that target immune checkpoint markers such as, for example, CTLA-4, LAG-3, and TIM-3, which can be targeted in a therapeutic method. Anti-PD-1 / PD-L1 immunotherapies include, but are not limited to, those therapies that comprise administering to a subject an effective amount of one or more anti-PD-1 / PD-L1 therapeutic antagonists, including, but not limited to, OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), Tecentriq™ (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242, etc. These antibodies may be administered as combination therapy with the anti-ABCC1 antibodies provided herein.

[0197] CTLA-4, also known as CD152, binds to CD80 and CD86. Antibodies against CTLA-4 have been approved for the treatment of several cancer types. The co-inhibitory effect of CTLA-4 with other immunotherapies makes CTLA-4 an excellent candidate for use in combination with other immunotherapies to treat certain cancers. TIM-3 can also be targeted for immunotherapy in some cancer types.

[0198] LAG-3 is currently undergoing clinical trials for the treatment of cancer. Anti-LAG-3 immunotherapy includes the use of antagonist LAG-3 antibodies that can both activate T effector cells (by downregulating LAG-3 inhibitory signals in preactivated LAG-3+ cells) and inhibit induced (i.e., antigen-specific) Treg suppressive activity. Useful LAG-3 antagonist antibodies include leratolimab (BMS-986016, developed by Bristol-Myers Squibb), IMP701 (developed by Immutep), and TSR-033 (anti-LAG-3 mAb, developed by TESARO, Inc.).

[0199] Immunotherapy also includes T cell-based immunotherapies, such as adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T cell therapy. For example, a subject may be administered a population of CAR T cells engineered to target an antigen expressed by the subject's cancer. T cell-based therapy, in some cases, may involve obtaining a cell sample, such as a blood sample or tumor biopsy, from the subject and culturing immune cells from the sample ex vivo, with or without genetic modification of the cultured immune cells. For example, immune cells may be obtained from the subject, cultured ex vivo, and engineered with a CAR specific to an antigen expressed by the cancer to produce a population of CAR T cells. The CAR T cells may then be reintroduced into the subject to target the cancer. T cell-based immunotherapy may be configured in various ways, such as by targeting different antigens or by collecting / culturing different cell types, depending on the particular cancer being treated. In addition, T cell-based immunotherapies can be administered systemically, e.g., by intravenous injection, or locally, e.g., by infusion (e.g., intraperitoneal infusion, pleural catheter infusion, etc.), direct injection, etc.

[0200] In some cases, the therapeutic methods described herein may include administering to a subject one or more inhibitors of a multidrug resistance transporter, including, but not limited to, a multidrug resistance transporter other than ABCC1. Useful inhibitors of multidrug resistance transporters include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and small molecule inhibitors. Inhibitors of multidrug resistance transporters include ABC transporter inhibitors.

[0201] Individuals suitable for treatment using the methods of the present disclosure include individuals who have cancer, individuals who have been diagnosed with cancer, individuals who are being treated for cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc., individuals who have been treated for cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) but have not responded to treatment, and individuals who have been treated for cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) and initially responded to treatment but subsequently relapsed, i.e., their cancer has recurred.

[0202] The methods of the present disclosure can be used to target and treat a variety of cancers, including, for example, primary cancers, secondary cancers, regrowing cancers, recurrent cancers, refractory cancers, etc. For example, in some cases, the methods of the present disclosure can be used as an initial treatment for a primary cancer identified in a subject. In some cases, the methods of the present disclosure can be used as a non-primary (e.g., second-line or later) treatment, for example, in subjects with cancer that is refractory to a previous treatment, in subjects with cancer that is regrowing after a previous treatment, in subjects with a mixed response to a previous treatment (e.g., a positive response to at least one tumor in the subject and a negative or neutral response to at least one second tumor in the subject), etc.

[0203] In some cases, the methods of the present disclosure can be used to treat subjects with drug-resistant cancers, such as multidrug-resistant cancers. Multidrug resistance (MDR) is the mechanism by which many cancers develop resistance to chemotherapeutic agents, resulting in minimal cell death and the expansion of drug-resistant tumors. MDR cancers can involve one or more resistance mechanisms, including, but not limited to, increased expression of efflux pumps, reduced absorption of drugs, inhibition of cell death or apoptosis, modulation of drug metabolism, etc. In some cases, the methods of the present disclosure can prevent, reverse, or circumvent MDR.

[0204] In some cases, the methods of the present disclosure can include treating a subject having a cancer that is resistant to a first agent with an effective amount of a subject antibody described herein in combination with a second agent that is different from the first agent. For example, in some cases, the subject's cancer can be resistant to a first chemotherapy, and the subject can be treated by administering an effective amount of a subject antibody as described herein in combination with a second chemotherapy that is different from the first. Various combinations of first and second chemotherapeutic agents can be used, depending, for example, on the type of cancer being treated, the likelihood of developing resistance, etc.

[0205] Many cancers are known to develop drug resistance. For this and other reasons, the methods of the present disclosure may find use in the treatment of a variety of cancers, including, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendix cancer, astrocytoma, atypical teratogenic / rhabdoid tumor, basal cell carcinoma, bile duct cancer (extrahepatic), bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma, etc.), brain stem glioma, brain tumors (e.g., astrocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, etc.), and the like. , craniopharyngioma, epithelioma, etc.), breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumors, Burkitt's lymphoma, carcinoid tumors (e.g., pediatric, gastrointestinal, etc.), carcinoma of unknown primary, cardiac (heart) tumors, central nervous system (e.g., atypical teratogenic / rhabdoid tumors, embryonal tumors, germ cell tumors, lymphoma, etc.), cervical cancer, pediatric cancer, spinal cord tumors, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal (e.g., bile duct, extrahepatic, etc.), non-invasive Ductal carcinoma in situ (DCIS), germinal tumor, endometrial cancer, epithelioma, esophageal cancer, neuroepithelioma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), fibrous histiocytoma of bone (e.g., malignant osteosarcoma, etc.), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular carcinoma (hepatic pancreatic cancer, histiocytosis (e.g., Langerhans cell, etc.), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (e.g., pancreatic neuroendocrine tumors, etc.), Kaposi's sarcoma, kidney cancer (e.g., renal cell, Wilms' tumor, childhood kidney tumors, etc.), Langerhans cell histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), hairy cell, etc.), lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.),Lymphomas (e.g., AIDS-related, Burkitt's, cutaneous T-cell, Hodgkin's, non-Hodgkin's, primary central nervous system (CNS), etc.), macroglobulinemia (e.g., Waldenstrom's, etc.), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary, midline duct carcinoma with Nat gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia (e.g., chronic (CML)), myeloid leukemia (e.g., acute (AML)), myeloproliferative neoplasms (e.g., chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer Cancer) (e.g., lip, etc.), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer (e.g., epithelial, germ cell tumors, low-grade malignant tumors, etc.), pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillary tumors, sinus tumors, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell carcinoma of the renal pelvis and urethra, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g., Ewing's, Kaposi's, osteosarcoma, rhabdomyosarcoma, soft tissue, uterine, etc.), Sezary syndrome, skin cancer (e.g., childhood, melanoma, Merkel cell carcinoma, non-melanoma, etc.), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer (e.g., with unknown primary, metastatic, etc.), gastric (stomach) cancer, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureter and renal pelvis cancer, urethral cancer, uterine cancer (e.g., endometrium, etc.), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, etc.

[0206] In some cases, the treatment methods described herein can be performed on subjects who have previously undergone one or more conventional treatments.For example, in the case of oncology, in some cases, the methods described herein can be performed following conventional cancer treatments, including but not limited to conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc. In some cases, the methods described herein can be used when the subject is not responding to conventional therapy or is refractory to conventional therapy.In some cases, the methods described herein can be used when the subject is responding to conventional therapy.

[0207] In some cases, the methods of the present disclosure can be used to target, treat, or eliminate subjects with minimal residual disease (MRD) remaining after a previous cancer treatment. Targeting, treating, and / or eliminating MRD can be pursued using the methods regardless of whether the MRD is refractory to a previous treatment. In some cases, the methods of the present disclosure can be used to target, treat, and / or eliminate subjects with MRD after a determination that the MRD is refractory to a previous treatment or to one or more available treatment options other than treatment with a multispecific antibody described herein.

[0208] In some cases, the method can be used prophylactically for surveillance. For example, a subject in need thereof can be administered treatment with one or more of the antibodies described herein when the subject does not have detectable disease but is at risk of developing recurrent cancer, including, for example, drug-resistant cancer. In some cases, a prophylactic approach can be used when the subject is at particularly high risk of developing a primary cancer that is predicted to be drug-resistant or that is expected to become drug-resistant. In some cases, a prophylactic approach can be used when the subject has previously been treated for cancer and is at risk of recurrence or developing drug resistance.

[0209] In some cases, the methods of the present disclosure may involve analyzing the cancer for expression of one or more markers or therapeutic targets. For example, in some cases, the methods may involve analyzing a cancer sample from a subject to determine whether the cancer expresses ABCC1 above a predetermined threshold.

[0210] In some cases, whether a subject is treated with an antibody of the present disclosure may depend on the results of the ABCC1 expression assessment. For example, in some cases, if the cancer expresses ABCC1 at or above a predetermined threshold, then the subject may be treated with an anti-ABCC1 antibody of the present disclosure; if the cancer expresses ABCC1 below the predetermined threshold, then the subject may not be treated with an anti-ABCC1 antibody of the present disclosure.

[0211] Any convenient assay can be used to analyze ABCC1 levels, including, but not limited to, flow cytometry, nucleic acid-based assays (e.g., amplification, sequencing, etc.), cell cytometry, immunohistochemistry, etc. Any convenient biological sample can be used, including, but not limited to, cancer biopsy samples. Useful predetermined thresholds for assessing the expression of one or more markers and / or targets can be determined by any convenient and appropriate method, including comparing measured expression levels with a corresponding control. For example, in some cases, a useful predetermined threshold for the level of ABCC1 in a sample can correspond to the level of ABCC1 measured in reference cells, such as healthy / normal cells.

[0212] How to make it As summarized above, the methods of the present disclosure also include methods of making and / or identifying antibodies as described herein. The subject antibodies can be produced by any known method, e.g., conventional synthetic methods for protein synthesis, recombinant DNA methods, etc.

[0213] If the subject antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When a polypeptide is chemically synthesized, synthesis can proceed via liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is bound to an insoluble support, followed by sequential addition of the remaining amino acids to the sequence, is an example of a suitable method for chemically synthesizing the subject antibody. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing the subject antibody.

[0214] Standard recombinant methods can be used to produce the subject antibodies. For example, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. The expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences, and the collection and purification of the antibody.

[0215] Due to the degeneracy of the genetic code, a variety of nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be generated by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of an earlier prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for preparing substitution, deletion, and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, target polypeptide DNA is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template incorporating the oligonucleotide primer, encoding the selected modification within the target polypeptide DNA.

[0216] Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selectable markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance, kanamycin-resistance, or neomycin-resistance) to permit detection of those cells transformed with the desired DNA sequences.

[0217] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding an antibody of interest. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0218] Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, along with suitable vectors having expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization, among others.

[0219] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce polypeptides of the invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, HEK cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0220] Once synthesized (either chemically or recombinantly), whole antibodies, dimers thereof, individual light and heavy chains, or other forms of the subject antibodies (e.g., scFv, etc.) can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (See generally, Scopes, Protein Purification, Springer-Verlag, NY, (1982)). The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% or more pure, and free of contaminants such as cellular debris, macromolecules other than the subject antibody, etc.

[0221] kit Aspects of the present disclosure also include kits. Kits can include, for example, any combination of antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. described herein. A subject kit can include one or more of a subject antibody, a nucleic acid encoding the same, or cells containing a subject nucleic acid. Kits can be configured for a variety of purposes, including, for example, therapeutic kits (e.g., the kit can include an anti-ABCC1 antibody and one or more additional active agents, such as, for example, chemotherapy), kits for producing antibodies, kits for screening antibodies, etc.

[0222] Optional components of the kits may vary and include, for example, buffers, protease inhibitors, etc. Where the subject kits include a subject nucleic acid, the nucleic acid may also have restriction sites, multiple cloning sites, primer sites, etc. The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined in a single container, if desired.

[0223] In addition to the above components, the subject kits can include instructions for using the kit components to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present on the labeling of the container of the kit or its components (i.e., associated with the packaging or subpackaging) as a package insert. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, such as a compact disc read-only memory (CD-ROM), digital versatile disc (DVD), diskette, etc. In still other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the Internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be displayed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate. [Example]

[0224] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are the following experiments intended to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0225] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Reagents, cloning vectors, cells, and kits for the methods mentioned in or related to this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and repositories such as, for example, Addgene, Inc., American Type Culture Collection (ATCC).

[0226] Example 1: Generation of antibodies that specifically bind to cells expressing ABCC1 Materials and Methods antibody generation Wild-type (WT) human and cynomolgus monkey ABCC1 (full-length and truncated forms) were used to immunize mice or rats. Spleen and lymph node cells from vaccinated animals were fused with SP2 / 0 myeloma cells (hybridoma technology). Hybridoma supernatants were screened for the presence of anti-ABCC1 antibodies by flow cytometry. CDRs from selected mouse IgGs were cloned into a mammalian IgG1 backbone expression vector for full-length IgG1 antibody expression and production in HEK293 host cells via transfection using standard protocols, as described below.

[0227] Expression vector To generate antibody expression vectors, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with either the human IgG1 constant heavy chain or the human IgG1 kappa constant light chain, which had been previously inserted into the respective generic recipient expression vectors optimized for expression in mammalian cell lines. The genes to be expressed were cloned into the pCI-neo mammalian expression vector (Promega), which uses the full-length human cytomegalovirus (CMV) immediate-early promoter for high-level gene expression. The two antibody chains were cloned into two different vectors.

[0228] The N-terminal signal sequences from mouse IgG heavy and kappa light chains were used for secretory expression of the heavy and light chains, respectively. The signal peptides were cleaved during expression, leaving intact N-termini. In the Fab constructs, the C-terminus of the CH1 IgG1 constant region was fused to a 6xHis tag for purification.

[0229] mAb production Antibody constructs were expressed using polymer-based co-transfection of Expi293 cells (A14527, ThermoFisher) cells growing in suspension with mammalian expression vectors according to the manufacturer's recommendations.

[0230] Approximately 6 days after transfection, cells were harvested by centrifugation. Specifically, 1 μg of total coding DNA per ml of transfected culture was diluted in Opti-MEM® medium (Life Technologies) and incubated with Expifectamine reagent (Life Technologies) in the same medium for 20 minutes. The mixture was then added to Expi293® cells growing in suspension in Expi293® Expression Medium (Life Technologies) at 2.5 million cells / ml at 37°C with and covered with 8% CO2 in air. After 6 days, the medium containing the antibody construct was harvested by centrifugation.

[0231] mAb purification To purify antibody formats containing human Fc, 10 μl of MabSelect™ SuRe™ (GE Healthcare) per ml of supernatant was added to the collected medium and stirred overnight at 4° C. The next day, Protein A resin was applied to a 24-well filter plate using a vacuum manifold unit (Pall Lifesciences, USA). The resin was washed with PBS, and the antibody was eluted in 50 mM phosphate pH 3 and neutralized with 10x PBS pH 13.

[0232] Analytical Tests for mAb (GXII Reduced and Non-Reduced) The purity and monomer content of the final protein preparations were determined by high-throughput analysis on a Caliper LabChip GXII using the Protein Express LabChip Kit (Perkin-Elmer) as described by the manufacturer. The chip was automatically primed on the instrument with a polymer solution containing 0.2% SDS and a fluorescent dye. The destaining channel was filled with a polymer solution without SDS or dye. Briefly, reducing and non-reducing conditions of the protein were prepared by mixing a small amount of sample (2-5 μL) with Caliper sample buffer, with or without DDT. The samples were denatured at 75°C for 5 min, centrifuged at 2000 g for 3 min, and then run. Electropherograms were generated using LabChip GXII Touch software (Perkin-Elmer).

[0233] Analytical Testing for mAb (HPLC) The purity and monomer content of the final protein preparation were determined by high-throughput analysis on HPLC. Size-exclusion chromatography (SEC) was performed on an Infinity1260 Agilent HPLC system using an Advancebio SEC 300A 4.6x300mm, 2.7µm (p / n PL1580-5301) (Agilent Technologies). Injections were performed under isocratic elution conditions using a mobile phase of PBS, 400mM sodium chloride, pH 7.4, and detection was by absorbance at 280nm. Quantification was based on the relative areas of the detected peaks.

[0234] The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% or more pure, and are free of contaminants such as, for example, cellular debris, macromolecules other than the subject antibodies, etc.

[0235] Monoclonal antibody titration binding to KPC1 Binding titration of recombinant antibodies to KPC1 transfectants was performed by serial dilution of the antibody starting from approximately 666 nM. Diluted antibody in flow cytometry buffer was incubated with cells on ice for 30 minutes. After two washes with flow cytometry buffer, bound antibody was detected with PE-labeled F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) diluted 1:200 in flow cytometry buffer and incubated with cells on ice for 20 minutes. After two washes with flow cytometry buffer, fluorescence was measured on an Attune NxT flow cytometer. Data were analyzed using GraphPad Prism 8.0 software to determine EC50 values.

[0236] Cell binding assay Antibody binding to cells was assessed by flow cytometry. 293T cells stably transfected to express human or cynomolgus ABCC1 were washed once in flow cytometry buffer (PBS + 2% FBS + 0.02% sodium azide), resuspended in flow cytometry buffer at 2 × 10^6 cells / mL, and dispensed into 96-well microtiter plates at 0.1 mL / well. Recombinant antibodies were added to cells at 5 μg / mL for initial binding confirmation or serially diluted in flow cytometry buffer starting from 100 μg / mL. After incubating cells on ice for 30 minutes, the cells were washed twice with flow cytometry buffer. Bound antibodies were detected with PE-labeled F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) and assessed on an Attune NxT flow cytometer. EC50 was calculated to be the antibody concentration that produced half of the maximal response.

[0237] ABCC1 efflux assay HEK293T cells expressing human ABCC1 were washed several times and cultured at 1 x 10 cells per ml in phenol red-free DMEM. 6The cells were aliquoted into 96-well plates at a cell density of 100 cells / well in 50 μl aliquots. The cells were mixed with 50 μl of antibody and incubated at 37°C for 1.5 hours. The cells were then washed twice and finally resuspended in 200 ml of PBS. Calcein AM fluorescence was measured by flow cytometry.

[0238] Cytotoxicity assay The effect of anti-ABCC1 antibodies on vincristine cytotoxicity was evaluated in 293T cells stably transfected to express ABCC1. Cells were seeded at 5000 cells / well in 0.05 mL of assay medium (DMEM + 10% FBS) in white, flat-bottom 96-well tissue culture plates. Vincristine was prepared at 2x the final assay concentration by serial dilution from 200 μM into assay medium containing test or control antibodies at 100 μg / mL (2x final concentration). An equal volume (0.05 mL) of the vincristine / antibody mixture was added to the 293T_ABCC1 cells in the 96-well plate. The plate was then incubated at 37°C in 5% CO2. After approximately 72–96 h, the plate was equilibrated to room temperature, and cell viability was assessed using the Promega® CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's recommended protocol. Luminescence was measured on a Molecular Devices® FlexStation® 3 multimode microplate reader and analyzed using GraphPad Prism 8.0 software. The half-maximal inhibitory concentration (IC50) is the concentration of drug (vincristine or other chemotherapeutic cytotoxic agent) at which the response (cell growth) is reduced by 50%.

[0239] CT26 syngeneic xenograft mouse model Syngeneic models are allografts immortalized from mouse cancer cell lines and then transplanted back into the same inbred immunocompetent mouse strain. CT26 is an N-nitroso-N-methylurethane (NNMU)-induced undifferentiated fibroblastic colon cancer cell line established from BALB / c mice with aggressive colon cancer. CT26 cells were purchased from ATCC® (CRL-2638™) and maintained at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. The cell line used was confirmed to be authentic and mycoplasma-negative. The cells adhere with a fibroblastic morphology and form tumors and metastases after transplantation into syngeneic BALB / c or immunodeficient mice.

[0240] 1 x 10 diluted in PBS:Matrigel (1:1) 6 Cells were implanted subcutaneously under sterile conditions using a 27G insulin syringe into anesthetized 5-6 week-old female Balb / c mice. All animal maintenance, handling, surveillance, and procedures were performed in accordance with approved APLAC protocols.

[0241] Tumor 100-150mm 3 Once this was reached, the mice were randomized into 6 groups of 5 mice each. 1. Control isotype 3 mg / kg, 2. Control isotype 3 mg / kg + doxorubicin 2 mg / kg 3. KNJY C1-831 3mg / kg 4. KNJY C1-831 3mg / kg + doxorubicin 5.KNJY C1-787A 3mg / kg 6. KNJY C1-787A 3mg / kg + doxorubicin 2mg / kg

[0242] Doxorubicin is soluble in water. All test articles were administered intraperitoneally twice a week for two consecutive weeks. Antibodies were administered 4 hours before doxorubicin.

[0243] Tumors were measured three times a week using calibrated calipers, and tumor volumes were calculated according to the formula 1 / 2*L*S*S, where L is the long axis and S is the short axis of the tumor. Body weights were recorded before treatment began and continuously monitored throughout the study. To avoid animal suffering, animals were euthanized when they became moribund according to the predefined criteria above: rapid weight loss, loss of ambulatory ability, respiratory distress, or inability to drink or eat.

[0244] result Table 3 lists the following characteristics of anti-ABCC1 antibodies: binding to 293T cells stably transfected to express human ABCC1 as measured by FACS, and binding to 293T cells stably transfected to express cynomolgus monkey ABCC1 as measured by FACS. [Table 3]

[0245] Figure 1 shows the results of a titration of 10 anti-ABCC1 monoclonal antibodies binding to the doxorubicin-resistant lung cancer cell line H69AR (ATCC® CRL-11351™), which endogenously expresses ABCC1, in the flow cytometry (FACS) assay described above. All antibodies tested show significant (>10-fold background fluorescence mean intensity (FMI)) binding to H69AR cells.

[0246] 2A-2B show the results of titration of the indicated anti-ABCC1 monoclonal antibodies to rat C6 glioma cell lines overexpressing human and cynomolgus ABCC1 in the FACS assay described above. All antibodies tested show significant binding to both human and cynomolgus ABCC1-overexpressing C6 cells.

[0247] Figures 3A-3C, 4, 5A-5B, and 6A-6B show the results of titration of additional anti-ABCC1 monoclonal antibodies to rat C6 glioma cell lines overexpressing human and cynomolgus ABCC1 in the FACS assay described above. "Second Ab only" refers to a non-primary antibody used as a negative control. All tested anti-ABCC1 antibodies show significant binding to both human ABCC1-overexpressing C6 cells and cynomolgus ABCC1-overexpressing C6 cells compared to the negative control.

[0248] 7A-7B show the results of an ABCC1 efflux assay performed using HEK293T cells expressing human ABCC1. All tested anti-ABCC1 antibodies significantly inhibit the efflux function of the ABCC1 transporter.

[0249] Figures 8A-8C show the titration binding and efflux assay characterization of humanized anti-ABCC1 monoclonal antibodies. Humanized anti-ABCC1 antibodies C1.831.hu11, C1.861.hu11, C1.861.hu21, and C1.844.hu21 bind to both human and cynomolgus monkey ABCC1 in titration binding assays and significantly inhibit the efflux function of the ABCC1 transporter in efflux assays.

[0250] 9A-9C show the binding of humanized variants of anti-ABCC1 monoclonal antibodies C1.831, C1.861, and C1.844 to rat C6 glioma cell lines overexpressing human and cynomolgus ABCC1. All humanized antibodies retain the ability to bind to both human and cynomolgus ABCC1.

[0251] 10A-10B show the binding of humanized anti-ABCC1 antibodies C1.851.12, C1.851.14, and C1.851.15 to the rat C6 glioma cell line overexpressing human and cynomolgus ABCC1. All three antibodies retain the ability to bind to both human and cynomolgus ABCC1.

[0252] Figures 11A-11C show the binding of four humanized ABCC1 / KT9 bispecific antibodies to human and cynomolgus monkey C6 cell lines overexpressing ABCC1 and KT9, respectively. Schematic bispecific antibody structures are also shown. KT9 represents the anti-PD-L1 monoclonal antibody atezolizumab. The bispecific antibodies contain the heavy and light chains from the indicated ABCC1 antibody and the scFv region formed from the KT9 antibody. All bispecific antibodies tested bind to both ABCC1-overexpressing C6 cells and KT9-overexpressing C6 cells.

[0253] Figures 12A-12C show the binding of four humanized C1 / KT1 bispecific antibodies to 293T cells expressing human ABCC1 and 293T cells expressing human or cynomolgus monkey KT1, respectively. KT1 represents the anti-ErbB2 (anti-HER2) monoclonal antibody trastuzumab. The bispecific antibodies contain the heavy and light chains from the indicated anti-ABCC1 antibodies and the scFv region formed from the KT1 antibody. The bispecific antibodies tested bind to both 293T cells expressing human KT1 and 293T cells expressing cynomolgus monkey KT1 and retain the ability to bind to 293T cells expressing human ABCC1.

[0254] Figures 13A-13B, 14A-14B, and 15 show the effect of tested anti-ABCC1 monoclonal antibodies on vincristine cytotoxicity in a 293T cytotoxicity assay. The tested anti-ABCC1 antibodies increase the cytotoxicity of vincristine in this assay. MK571 is a commercially available small molecule inhibitor of ABCC1-mediated transport.

[0255] Figure 16 shows that the three tested anti-ABCC1 monoclonal antibodies inhibit tumor growth in vivo in the H69AR cytotoxicity assay, which evaluates the effect of the tested antibodies on vincristine cytotoxicity of the H69AR cell line, an adriamycin-selected C1-positive variant of the human small cell lung cancer cell line, NCI-H69.

[0256] FIG. 17 shows that anti-ABCC1 monoclonal antibodies C1-831 and C1-737A inhibit tumor growth in vivo in the CT26 syngeneic mouse tumor model.

[0257] The present disclosure includes the following embodiments. Embodiment 1 1. An antibody that specifically binds to ATP-binding cassette subfamily C member 1 (ABCC1) on the surface of a mammalian cell, wherein the antibody is characterized in that, for binding to ABCC1, An antibody that competes with an antibody comprising heavy chain complementarity-determining regions 1 to 3 (HCDR1 to 3) and light chain CDR1 to 3 (LCDR1 to 3) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. Embodiment 2 The antibody of embodiment 1, wherein the antibody comprises HCDRs 1 to 3 of the VH region of an antibody listed in Table 2. Embodiment 3 The antibody of embodiment 2, wherein the antibody comprises LCDRs 1 to 3 of the VL region of an antibody listed in Table 2. Embodiment 4 The antibody, The antibody of embodiment 1, comprising the heavy chain complementarity determining regions (HCDRs) and light chain CDRs (LCDRs) of the paired variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. Embodiment 5 An antibody molecule that specifically binds to ATP-binding cassette subfamily C member 1 (ABCC1) on the surface of mammalian cells. Embodiment 6 The antibody (a) a variable heavy chain (VH) region comprising heavy chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) of the VH region of an antibody listed in Table 2; (b) a variable light chain (VL) region comprising the light chain CDRs 1-3 (LCDRs 1-3) of the VL region of an antibody listed in Table 2; (c) a VH region comprising HCDRs 1 to 3 of the VH region of an antibody listed in Table 2, and a VL region comprising LCDRs 1 to 3 of the VL region of an antibody listed in Table 2; or (d) The antibody molecule of embodiment 5, comprising a VH region comprising HCDRs 1 to 3 of the VH region of an antibody listed in Table 2, and a VL region comprising LCDRs 1 to 3 of the VL region of said antibody. Embodiment 7 The antibody molecule of embodiment 6, wherein the antibody comprises HCDRs 1-3 and LCDRs 1-3 of a pair of VH and VL regions of an antibody listed in Table 2. Embodiment 8 The antibody of embodiment 6, wherein the antibody comprises HCDRs 1-3 of the VH region of a first antibody listed in Table 2. Embodiment 9 The antibody molecule of embodiment 8, wherein the antibody comprises LCDRS1-3 of the VL region of a second antibody in Table 2. Embodiment 10 The antibody molecule of embodiment 6, wherein the antibody molecule comprises the variable light (VL) chain and / or the variable heavy (VH) chain of an antibody listed in Table 2. Embodiment 11 The antibody i. a variable heavy chain (VH) region comprising heavy chain complementarity determining regions 1-3 (HCDRs 1-3) of the VH region of a C1.831.hu41 antibody, a C1.831.hu11 antibody, a C1.844.hu21 antibody, a C1.851.hu15 antibody, a C1.851.hu12 antibody, or a C1.861.hu11 antibody listed in Table 2; ii. a variable light chain (VL) region comprising light chain CDRs 1-3 (LCDRs 1-3) of the VL region of a C1.831.hu41 antibody, a C1.831.hu11 antibody, a C1.844.hu21 antibody, a C1.851.hu15 antibody, a C1.851.hu12 antibody, or a C1.861.hu11 antibody listed in Table 2; iii. a VH region comprising HCDRs 1-3 of the VH region of a C1.831.hu41 antibody, a C1.831.hu11 antibody, a C1.844.hu21 antibody, a C1.851.hu15 antibody, a C1.851.hu12 antibody, or a C1.861.hu11 antibody listed in Table 2, and a VL region comprising LCDRs 1-3 of the VL region of a C1.831.hu41 antibody, a C1.831.hu11 antibody, a C1.844.hu21 antibody, a C1.851.hu15 antibody, a C1.851.hu12 antibody, or a C1.861.hu11 antibody listed in Table 2; iv. a VH region comprising HCDRs 1-3 and a VL region comprising LCDRs 1-3 of the VH region and VL region of the C1.831.hu41 antibody, C1.831.hu11 antibody, C1.844.hu21 antibody, C1.851.hu15 antibody, C1.851.hu12 antibody, or C1.861.hu11 antibody, respectively, listed in Table 2; or v. The antibody molecule of embodiment 5, comprising the VH and VL regions of the C1.831.hu41 antibody, the C1.831.hu11 antibody, the C1.844.hu21 antibody, the C1.851.hu15 antibody, the C1.851.hu12 antibody, or the C1.861.hu11 antibody listed in Table 2. Embodiment 12 The antibody i. a VH region comprising HCDRs 1 to 3 of the VH region of the C1.830B.hu11 antibody, C1.851.hu11 antibody, C1.851.hu13 antibody, C1.787a.hu11 antibody, C1.844.hu11 antibody, C1.861.hu21 antibody, C1.861.hu41 antibody, C1.861.hu61 antibody, or C1.851.hu14 antibody listed in Table 2; ii. a VL region comprising LCDRs 1 to 3 of the VL region of the C1.830B.hu11 antibody, C1.851.hu11 antibody, C1.851.hu13 antibody, C1.787a.hu11 antibody, C1.844.hu11 antibody, C1.861.hu21 antibody, C1.861.hu41 antibody, C1.861.hu61 antibody, or C1.851.hu14 antibody listed in Table 2; iii. A VH region comprising HCDRs 1 to 3 of the VH region of the C1.830B.hu11 antibody, C1.851.hu11 antibody, C1.851.hu13 antibody, C1.787a.hu11 antibody, C1.844.hu11 antibody, C1.861.hu21 antibody, C1.861.hu41 antibody, C1.861.hu61 antibody, or C1.851.hu14 antibody listed in Table 2. and a VL region comprising LCDR1 to LCDR3 of the VL region of the C1.830B.hu11 antibody, C1.851.hu11 antibody, C1.851.hu13 antibody, C1.787a.hu11 antibody, C1.844.hu11 antibody, C1.861.hu21 antibody, C1.861.hu41 antibody, C1.861.hu61 antibody, or C1.851.hu14 antibody listed in Table 2; iv. a VH region comprising HCDRs 1-3 and a VL region comprising LCDRs 1-3 of the VH region and VL region of the C1.830B.hu11 antibody, C1.851.hu11 antibody, C1.851.hu13 antibody, C1.787a.hu11 antibody, C1.844.hu11 antibody, C1.861.hu21 antibody, C1.861.hu41 antibody, C1.861.hu61 antibody, or C1.851.hu14 antibody, respectively, listed in Table 2; or v. The antibody molecule of embodiment 5, comprising the VH and VL regions of the C1.830B.hu11 antibody, C1.851.hu11 antibody, C1.851.hu13 antibody, C1.787a.hu11 antibody, C1.844.hu11 antibody, C1.861.hu21 antibody, C1.861.hu41 antibody, C1.861.hu61 antibody, or C1.851.hu14 antibody listed in Table 2. Embodiment 13 The antibody i. a VH region comprising HCDRs 1 to 3 of the VH region of a C1.773 antibody, a C1.773a antibody, a C1.777a antibody, a C1.784a antibody, a C1.786a antibody, a C1.787a antibody, a C1.827 antibody, a C1.830B antibody, a C1.831 antibody, a C1.835 antibody, a C1.841 antibody, a C1.844 antibody, a C1.845 antibody, a C1.847 antibody, a C1.851 antibody, a C1.855 antibody, a C1.861 antibody, a C1.863 antibody, a C1.876 antibody, a C1.877 antibody, or a C1.879A antibody listed in Table 2; ii. a VL region comprising LCDRs 1 to 3 of the VL region of a C1.773 antibody, a C1.773a antibody, a C1.777a antibody, a C1.784a antibody, a C1.786a antibody, a C1.787a antibody, a C1.827 antibody, a C1.830B antibody, a C1.831 antibody, a C1.835 antibody, a C1.841 antibody, a C1.844 antibody, a C1.845 antibody, a C1.847 antibody, a C1.851 antibody, a C1.855 antibody, a C1.861 antibody, a C1.863 antibody, a C1.876 antibody, a C1.877 antibody, or a C1.879A antibody listed in Table 2; iii. A VH region comprising HCDRs 1 to 3 of the VH region of a C1.773 antibody, a C1.773a antibody, a C1.777a antibody, a C1.784a antibody, a C1.786a antibody, a C1.787a antibody, a C1.827 antibody, a C1.830B antibody, a C1.831 antibody, a C1.835 antibody, a C1.841 antibody, a C1.844 antibody, a C1.845 antibody, a C1.847 antibody, a C1.851 antibody, a C1.855 antibody, a C1.861 antibody, a C1.863 antibody, a C1.876 antibody, a C1.877 antibody, or a C1.879A antibody listed in Table 2. and a VL region comprising LCDR1 to LCDR3 of the VL region of the C1.773 antibody, C1.773a antibody, C1.777a antibody, C1.784a antibody, C1.786a antibody, C1.787a antibody, C1.827 antibody, C1.830B antibody, C1.831 antibody, C1.835 antibody, C1.841 antibody, C1.844 antibody, C1.845 antibody, C1.847 antibody, C1.851 antibody, C1.855 antibody, C1.861 antibody, C1.863 antibody, C1.876 antibody, C1.877 antibody, or C1.879A antibody listed in Table 2; iv. a VH region comprising HCDRs 1 to 3 and a VL region comprising LCDRs 1 to 3 of the VH region and VL region of a C1.773 antibody, a 773a antibody, a 777a antibody, a 784a antibody, a 786a antibody, a 787a antibody, a C1.827 antibody, a C1.830B antibody, a C1.831 antibody, a C1.835 antibody, a C1.841 antibody, a C1.844 antibody, a C1.845 antibody, a C1.847 antibody, a C1.851 antibody, a C1.855 antibody, a C1.861 antibody, a C1.863 antibody, a C1.876 antibody, a C1.877 antibody, or a C1.879A antibody, each of which is listed in Table 2; or v. The antibody molecule of embodiment 5, comprising the VH and VL regions of a C1.773 antibody, a 773a antibody, a 777a antibody, a 784a antibody, a 786a antibody, a 787a antibody, a C1.827 antibody, a C1.830B antibody, a C1.831 antibody, a C1.835 antibody, a C1.841 antibody, a C1.844 antibody, a C1.845 antibody, a C1.847 antibody, a C1.851 antibody, a C1.855 antibody, a C1.861 antibody, a C1.863 antibody, a C1.876 antibody, a C1.877 antibody, or a C1.879A antibody listed in Table 2. Embodiment 14 The antibody molecule of any one of embodiments 11 to 13, wherein the antibody comprises a human IgG Fc region. Embodiment 15 The antibody molecule of any one of embodiments 11 to 13, wherein the antibody comprises a human IgG1 Fc region. Embodiment 16 The antibody molecule of any one of embodiments 11 to 13, wherein the antibody comprises a human IgG1 constant heavy chain and constant light chain region. Embodiment 17 2. The antibody molecule of any one of the preceding embodiments, wherein said antibody, when bound to a cell expressing ABCC1, inhibits efflux by said ABCC1. Embodiment 18 10. The antibody molecule of any one of the preceding embodiments, wherein the antibody comprises a humanized light chain. Embodiment 19 The antibody molecule of any one of the preceding embodiments, wherein the antibody comprises a humanized heavy chain. Embodiment 20 The antibody may be a bispecific antibody, an Ig monomer, a Fab fragment, a F(ab') 2 10. The antibody molecule of any one of the preceding embodiments, wherein the antibody molecule is selected from the group consisting of: a Fd fragment, an scFv, an scAb, a dAb, and an Fv. Embodiment 21 The antibody molecule of any one of the preceding embodiments, wherein the antibody is a bispecific antibody comprising the VH region and VL region of any one of embodiments 1 to 20, and further comprising a second VH region comprising HCDRs 1 to 3 of an antibody that binds to a tumor-associated antigen (TAA). Embodiment 22 The antibody molecule of embodiment 21, wherein the antibody comprises a second VL region, and the second VH region and the second VL region bind to the TAA. Embodiment 23 23. The antibody molecule of embodiment 22, wherein the second VH region and the second VL region are present in a single polypeptide. Embodiment 24 The antibody molecule of embodiment 22, wherein the second VH region and the second VL region are present in an scFv. Embodiment 25 The antibody molecule of embodiment 21, wherein the antibody comprises a common light chain, and the common light chain comprises a VL region of any one of embodiments 1 to 20. Embodiment 26 The antibody molecule of any one of embodiments 21 to 25, wherein said TAA is PD-L1. Embodiment 27 27. The antibody molecule of embodiment 26, wherein the antibody that binds to PD-L1 is atezolizumab. Embodiment 28 28. The antibody molecule of embodiment 27, wherein the bispecific antibody comprises a VH region comprising HCDRs 1 to 3 of the VH region and a VL region comprising LCDRs 1 to 3 of the VH region and VL region of the C1.844 antibody or the C1.851 antibody listed in Table 2, respectively, and an scFv comprising HCDRs 1 to 3 and LCDRs 1 to 3 of atezolizumab. Embodiment 29 28. The antibody molecule of embodiment 27, wherein the bispecific antibody comprises the VH and VL regions of the C1.844hu21 or C1.851hu12 antibody listed in Table 2 and an scFv comprising HCDRs 1-3 and LCDRs 1-3 of atezolizumab. Embodiment 30 The TAA ErbB2 (HER2), an antibody molecule according to any one of embodiments 21 to 25. Embodiment 31 The antibody molecule of embodiment 30, wherein said antibody that binds to ErbB2 is trastuzumab. Embodiment 32 The antibody molecule of embodiment 31, wherein the bispecific antibody comprises a VH region comprising HCDRs 1 to 3 of the VH region and a VL region comprising LCDRs 1 to 3 of the VH region and VL region of the C1.844 antibody, the C1.831 antibody, or the C1.851 antibody listed in Table 2, respectively, and an scFv comprising HCDRs 1 to 3 and LCDRs 1 to 3 of trastuzumab. Embodiment 33 32. The antibody molecule of embodiment 31, wherein the bispecific antibody comprises the VH and VL regions of the C1.844hu21 antibody, C1.831hu11 antibody, or C1.851hu12 antibody listed in Table 2, and an scFv comprising HCDRs 1-3 and LCDRs 1-3 of trastuzumab. Embodiment 34 The antibody molecule of any one of embodiments 1 to 33, wherein the antibody comprises a VL region and a VH region that are present in separate polypeptides. Embodiment 35 The antibody molecule of any one of embodiments 1 to 33, wherein the antibody comprises a VL region and a VH region present in a single polypeptide. Embodiment 36 36. The antibody molecule of any one of embodiments 1 to 35 for use in a method of treating cancer in a subject, said method comprising administering said antibody to said subject. Embodiment 37 37. The antibody molecule for use of embodiment 36, wherein the method comprises administering the antibody in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. Embodiment 38 38. The antibody molecule for use of embodiment 37, wherein said at least one additional active agent is a chemotherapeutic agent, optionally wherein said chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate. Embodiment 39 The antibody molecule for use according to any one of embodiments 36 to 38, wherein the subject being treated has a cancer that has been determined to be resistant to treatment with said chemotherapeutic agent. Embodiment 40 1. A pharmaceutical composition comprising: an antibody of any one of the preceding embodiments; and and a pharmaceutically acceptable excipient. Embodiment 41 41. The pharmaceutical composition of embodiment 40, further comprising an additional active agent. Embodiment 42 The pharmaceutical composition of embodiment 41, wherein the additional active agent is a chemotherapeutic agent. Embodiment 43 42. The pharmaceutical composition of embodiment 41, wherein the additional active agent comprises an inhibitor of a multidrug resistance transporter. EMBODIMENT 44 The pharmaceutical composition of embodiment 41, wherein the additional active agent comprises an immunotherapeutic agent. Embodiment 45 One or more nucleic acids comprising one or more sequences encoding the antibody molecule of any one of embodiments 1 to 35. Embodiment 46 One or more recombinant expression vectors comprising one or more nucleic acids of embodiment 45. Embodiment 47 A host cell genetically modified with one or more recombinant expression vectors of embodiment 46. Embodiment 48 An immune effector cell comprising a chimeric antigen receptor (CAR) comprising an ABCC1-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the ABCC1-binding domain comprises heavy chain complementarity determining regions 1-3 (HCDR1-3) of a variable heavy chain (VH) region of an antibody listed in Table 2, and / or light chain CDR1-3 (LCDR1-3) of a variable light chain (VL) region of an antibody listed in Table 2. Embodiment 49 36. A method for assaying expression of ABCC1 on the cell surface of a cell, comprising contacting said cell with the antibody of any one of embodiments 1 to 35. Embodiment 50 50. The method of embodiment 49, wherein the antibody is detectably labeled. Embodiment 51 36. A method for inhibiting the efflux activity of ABCC1 expressed by a living cell, the method comprising contacting said cell with the antibody of any one of embodiments 1 to 35. Embodiment 52 52. The method of embodiment 51, further comprising contacting the cells with an inhibitor of ABCC1-mediated efflux. Embodiment 53 53. The method of embodiment 51 or 52, further comprising contacting the cells with a chemotherapeutic agent. EMBODIMENT 54 54. The method of any one of embodiments 51 to 53, wherein the cells are cancer cells. Embodiment 55 55. The method of embodiment 54, wherein the cancer cells are multidrug resistant cancer cells. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0258] Thus, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to furthering this technology, and should be construed as not being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly recited in the claims.

[0259] Accordingly, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined as being invoked for a claim limitation only if the precise phrase "means for" or the precise phrase "step for" appears at the beginning of such claim limitation; if such precise phrases are not used in the claim limitation, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

Claims

1. A bispecific antibody molecule that specifically binds to ATP-binding cassette subfamily C member 1 (ABCC1) and a tumor-associated antigen (TAA) on the surface of a mammalian cell, comprising: (i) the TAA is PD-L1 and the bispecific antibody comprises the VH and VL regions of the C1.844hu21 or C1.851hu12 antibody listed in Table 2 and an scFv comprising HCDR1-3 and LCDR1-3 of atezolizumab; or (ii) the TAA is ErbB2 (HER2), and the bispecific antibody comprises the VH and VL regions of the C1.844hu21 antibody, C1.831hu11 antibody, or C1.851hu12 antibody listed in Table 2, and an scFv comprising HCDR1-3 and LCDR1-3 of trastuzumab; Bispecific antibody molecule.

2. The bispecific antibody molecule of claim 1 , wherein the bispecific antibody comprises a VL region and a VH region that are present in separate polypeptides.

3. The bispecific antibody molecule of claim 1 , wherein the bispecific antibody comprises a VL region and a VH region present in a single polypeptide.

4. 10. A composition for use in a method of treating cancer in a subject, said composition comprising a bispecific antibody molecule according to any one of claims 1 to 3, said method comprising administering said bispecific antibody to said subject.

5. 5. The composition of claim 4, wherein the method comprises administering the bispecific antibody in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof.

6. The composition of claim 5 , wherein the at least one additional active agent is a chemotherapeutic agent.

7. The chemotherapeutic agent (ii) taxol, vinca alkaloids, anthracyclines, etoposide, mitoxantrone, or methotrexate; or (iii) a metal complex; The composition of claim 6.

8. The composition of claim 7, wherein the metal complex is cisplatin or carboplatin.

9. The composition of any one of claims 4 to 8, wherein the subject being treated has a cancer that has been determined to be resistant to treatment with the chemotherapeutic agent.

10. One or more nucleic acids comprising one or more sequences encoding the bispecific antibody molecule of any one of claims 1 to 3.

11. 11. One or more recombinant expression vectors comprising one or more nucleic acids of claim 10.

12. A host cell genetically modified with one or more recombinant expression vectors of claim 11.

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