Anti- ABCC1 antibodies and uses thereof

Abstract

To provide anti- ABCC1 antibodies and uses thereof.SOLUTION: Antibodies that target the efflux pump ATP-binding cassette subfamily C member 1 (ABCC1) are provided. Also provided are pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits comprising or encoding such antibodies. Also disclosed are 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. Also provided are methods for treating a subject for ABCC1 comprising administering to the subject the anti-cancer antibodies disclosed herein. A subject antibody can be a bispecific antibody. Bispecific antibodies may bind ABCC1 and tumor-associated antigens (TAA).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 073826, filed September 2, 2020. The benefit of priority is claimed, and that application is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listings provided as text files The sequence listing was created on August 24, 2021, and is a text file with a size of 151 KB. This specification is stored in the file "KNJY-006WO SEQ LIST_ST25.txt". The contents of the text file are incorporated herein by reference in their entirety. Be absorbed.

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

[0004] Efflux pumps (EPs) are proteins expressed by living cells that transport a variety of compounds. They have evolved to be naturally exported from cells. Members of the Tractor family of proteins are examples of EPs that enable drug efflux. The structure of transporters varies from protein to protein (e.g., there are 49 known ABC transporters in humans). family members), they all have two distinct domains - the highly conserved nucleotide They are classified by the presence of a nucleotide-binding domain and a more variable transmembrane domain. Encoded by the TP-binding cassette subfamily B member 1 (ABCB1) gene The multidrug resistance protein 1 (MDR1), which is responsible for the ATP-binding cassette subfamily C member 1 (AB CC1) expression increases in response to treatment with certain chemotherapeutic agents.

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

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

[0007] Targeting the cellular efflux pump ATP-binding cassette subfamily C member 1 (ABCC1) Pharmaceutical compositions, nucleic acids, recombinant proteins, and methods for the preparation of such antibodies are provided. Expression vectors, cells, and kits are also provided. for detecting the presence or absence of BCC1 expression, the level of ABCC1 expression, and / or Also disclosed are methods of using the antibodies to inhibit ABCC1 function. treating a subject for cancer, comprising administering an anti-ABCC1 antibody disclosed herein. A method for doing so is 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" refer to an antibody or immunoglobulin of any isotype. globulin, Fab, Fv, scFv, Fd, Fab', Fv, and F(ab')2, Antibody fragments that retain specific binding to an antigen include, but are not limited to, chimeric antibodies, human antibodies, and the like. Antibodies, including immobilized antibodies, monoclonal antibodies, and antibodies containing only heavy chains (e.g., VHH camelid antibodies) Single chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies may be used in combination with other antibodies, including, for example, radioisotopes, enzymes that produce detectable products, The antibody may be detectably labeled, such as with a fluorescent protein. , for example, to another moiety such as biotin (a member of the biotin-avidin specific binding pair). The antibody may also be conjugated to a polystyrene plate or bead, etc. The antibody may be attached to a solid support, including but not limited to, a nucleotide sequence ... The antibody may be conjugated to a toxic moiety, such as a chemotherapeutic agent.

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

[0011] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. The 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 of each variable domain interact to form a VH-VL pair. The six CDRs define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs provide the antigen-binding domain for the antibody. However, a single variable domain (or three domains specific for an antigen) Although even an Fv half containing only the CDRs of each variable region has the ability to recognize and bind to antigen, It has lower affinity than the entire binding site containing the three CDRs of the 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 contain the heavy chain CH1 domain containing one or more cysteines from the antibody hinge region. The addition of a few residues at the carboxyl terminus of the Fab fragment differentiates it from the Fab' fragment. Fab'-SH is a Fab fragment in which the cysteine ​​residues of the constant domains have free thiol groups. The F(ab')2 antibody fragment is the counterpart of the Fab' fragment. They were first produced with hinge cysteines between them. Other chemistries of antibody fragments Coupling is also known.

[0013] The "light chains" of antibodies (immunoglobulins) from any vertebrate species consist of their constant domains Based on the amino acid sequence, there are two distinct types of cytoplasm, called kappa and lambda. Depending on the amino acid sequence of the constant domain of their heavy chains, Immunoglobulins can be assigned to different classes by There are major classes: IgA, IgD, IgE, IgG, and IgM, of which Some are further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, It can be divided into IgG4, IgA, and IgA2.

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

[0015] The term "diabody" refers to a small antibody fragment that has two antigen-binding sites, This fragment contains a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same position. VH-VL polypeptide chains (VH-VL) that allow pairing between two domains on the same chain. By using a linker that is too short for the domain to be able to cleave the complementary domain on another strand, Diabodies are, for example, EP404,0 97, WO93 / 11161, and Hollinger et al., Proc. Na Acad. Sci. USA, 90:6444-6448 (1993) It is written.

[0016] As used herein, the term "affinity" refers to the ability of two agents to bind reversibly. The affinity is the equilibrium constant for the interaction of unrelated amino acid sequences and is expressed as the dissociation constant (Kd). At least 1-fold greater, at least 2-fold greater, or less than the affinity of the antibody for the column Both are 3 times larger, at least 4 times larger, at least 5 times larger, and at least 6 times larger , at least 7 times larger, at least 8 times larger, at least 9 times larger, at least 1 0 times larger, at least 20 times larger, at least 30 times larger, at least 40 times larger At least 50 times larger At least 60 times larger At least 70 times larger At least At least 80 times larger, at least 90 times larger, at least 100 times larger, or at least The target protein can be at least 1000 times larger, or even larger. The affinity of the antibody is, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about It can be 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the ability to dissociate after dilution. "Immunoreactive" and "preferentially binding" refer to the resistance of a complex of two or more drugs. The terms "antibody" and "antigen-binding fragment" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments. .

[0017] The term "binding" includes covalent interactions, electrostatic interactions, hydrophobic interactions, as well as e.g. For example, by ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. refers to a direct association between the two molecules. ABCC1-specific antibodies bind to the ABCC1 polypeptide. It specifically binds to an epitope within a given molecule. An epitope is formed by a continuous stretch of amino acids. a linear epitope formed by a non-contiguous stretch of amino acids, or a non-linear or steric epitope formed by a non-contiguous stretch of amino acids. Non-specific binding may be about 10 -7 Binding with an affinity less than M, e.g. For example, 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" refers to a heavy chain The term "antigen binding site" refers to the non-contiguous antigen binding sites found within the variable regions of both the light and dark chain polypeptides. It is intended that the CDRs are hypervariable regions, also called "framework regions (FR)." The CDRs are interspersed with more conserved regions, as shown by Kabat et al. , J. Biol. Chem. 252:6609-6616 (1977), Kabat e t al.,USDept.of Health and Human Servi ces,“Sequences of proteins of immunology cal interest”(1991), Chothia et al., J.Mol Biol.196:901-917(1987), and MacCallum et al. I., J. Mol. Biol. 262:732-745 (1996) The definition includes overlapping amino acid residues or their subunits when compared against each other. Nevertheless, the CDRs of antibodies or their grafted antibodies or The application of either definition to a variant is within the scope of the term as defined and used herein. It is intended to encompass the CDRs defined by each of the above cited documents. The amino acid residues that are present are shown in Table 1 below for comparison. [Table 1]

[0019] As used herein, the term "framework" refers to an antibody variable region. When used, it refers to all amino acid residues outside the CDR regions in the variable region of an antibody. It is contemplated that the variable region framework will generally be approximately 100-120 amino acids in length. A non-contiguous amino acid sequence of amino acids, but refers only to those amino acids outside the CDRs. As used herein, the term "framework region" means a is intended to mean each of the framework domains separated by the CDRs. The VH chain is arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, and may comprise three CDRs and four FRs arranged from the N-terminus to the C-terminus. Similarly, the VL chain has the following order: FR1, CDR1, FR2, CDR2, FR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR119, CDR12, CDR13, CDR14, C It contains three CDRs and four FRs, DR3 and FR4, arranged from the N-terminus to the C-terminus. 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 refers to a tetramer of two heavy chains and two light chains. The heavy and light chains are interconnected by, for example, disulfide bonds. The light chain constant region consists of three domains, CH1, CH2, and CH3. The variable regions of the heavy and light chains are the domains that interact with antigens. The constant region of an antibody typically contains the first receptors for various cells of the immune system and the complement system. The term "antibody" refers to a molecule that mediates the binding of antibodies to host tissues and factors, including components thereof. Immunoglobulin types IgA, IgG, IgE, IgD, IgM, and their In some embodiments, the subject antibody is of the IgG isotype, e.g., For example, IgG1.

[0021] As used herein, the term "immunoglobulin" refers to a gene encoding an immunoglobulin gene. It refers to a protein comprising one or more polypeptides substantially encoded by The human immunoglobulin genes identified include kappa, lambda, and alpha (IgA1 and IgA2) ), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mi The full length immunoglobulins include the ribosomal constant region genes, as well as many immunoglobulin variable region genes. The globulin light chain (approximately 25 kD or 214 amino acids) is composed of an N-terminal variable region gene (approximately The total length of the ribosomal protein is 110 amino acids, and the C-terminal kappa or lambda constant region. Long immunoglobulin heavy chains (approximately 50 kD or 446 amino acids) contain an N-terminal variable region gene a sequence of about 116 amino acids and one of the other constant region genes mentioned above at the C-terminus, e.g., For example, it is encoded by gamma (encoding approximately 330 amino acids). In some embodiments, the subject antibodies comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain. nothing.

[0022] The term "antigen-binding fragment" refers to a fragment of a full-length antibody that is capable of specifically binding to an antigen. Examples of binding fragments include (i) Fab fragments (VL, VH, CL, and CH) (ii) a monovalent fragment comprising, e.g., consisting of, one or more F(ab')2 domains; fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region) (iii) Fd fragment (comprising, e.g., consisting of, a VH and a CH1 domain) (iv) Fv fragments (e.g., comprising the VH and VL domains of a single arm of an antibody, e.g., (v) dAb fragments (including, e.g., consisting of, a VH domain) (vi) isolated CDRs; (vii) single-chain Fvs (scFvs) (e.g., VH and VL The domain pairs are linked by synthetic linkers using recombinant means to form monovalent molecules. comprising, for example consisting of, the VH and VL domains of a single arm of an antibody viii) diabodies (VH and VL domains are separated so that they do not form monovalent molecules) comprising, e.g., consisting of, two scFvs that are not paired, and each one of the scFvs One VH pairs with the VL domain of another scFv to form a bivalent molecule).

[0023] The term "chimeric" antibody refers to an antibody in which portions of the heavy and / or light chains are derived from a particular source or species. while the remainder of the heavy and / or light chains are derived from a different source or species. Refers to...

[0024] A "human antibody" is an antibody produced by a human or human cells, or derived from the human antibody repertoire. or other non-human sources utilizing human antibody coding sequences. This definition of a human antibody includes non-human antigen-binding residues. Humanized antibodies are expressly excluded.

[0025] A "human consensus framework" is a human immunoglobulin variable light (VL) or variable The most commonly occurring amino acid residues in the variable heavy (VH) framework sequence selection were Generally, the selection of a human immunoglobulin VL or VH sequence is The selection is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is abat et al., Sequences of Proteins of Imm unological interest,Fifth Edition,NIH Pu Blication 91-3242, Bethesda Md. (1991), vol. In one embodiment, for VL, The subgroup is kappa I as in Kabat et al. (supra). In an embodiment, for VH, the subgroups are as described in Kabat et al. (supra). This is subgroup III.

[0026] A "humanized" antibody is one that contains amino acid residues from non-human CDRs and human framework (FR) regions. A chimeric antibody refers to an antibody containing at least one amino acid residue from a humanized antibody constant region. The moiety is derived from a human antibody, e.g., a human IgG1 antibody. In a preferred embodiment, The antibody molecules disclosed herein comprise a variable heavy chain region as provided herein, and a UniPr Human Ig having the amino acid sequence shown in ot:P01857-1, version 1 In a preferred embodiment, the antibody disclosed herein comprises a heavy chain comprising a G1 constant region. The molecule comprises a light chain comprising a variable light chain region as provided herein and a human light chain constant region. In a preferred embodiment, the human light chain constant region is rot: a human kappa light chain constant region having the amino acid sequence described in P01834.2 In certain embodiments, the human IgG1 heavy chain constant region present in the subject antibody is mutated, For example, substitutions that modulate Fc function can be included, e.g., LALAPG effector function modifications. The mutations (L234A, L235A, and P329G) or N297A were introduced to enhance antibody-dependent The numbering of substitutions follows the EU numbering system. The "EU numbering system" or "EU index" is based on immunoglobulin heavy chain numbering. It is commonly used when referring to residues in the constant region (e.g., Kabat et al. .,Sequences of Proteins of Immunology Interest,5th Ed.Public Health Service,N ational Institutes of Health,Bethesda,MD (1991)). "Index" refers to the residue numbering of the human IgG 1 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 molecule recognized by the immune system, for example, by antibodies, B cells, or T cells. For example, an epitope is the specific region of an antigen to which an antibody binds. be.

[0029] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment may interfere with diagnostic or therapeutic uses for the antibody. materials that can contain enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody has: (1) a cytoplasmic affinity to the antibody as determined by the Lowry method; more than 90%, more than 95%, or more than 98% by weight of the antibody, for example, up to more than 99% by weight, (2 ) By using a spinning cup sequenator, a small amount of N-terminal or internal amino acid sequence can be obtained. (3) Coomassie blue or silver Staining using sodium dodecyl sulfate-polyacrylic acid under reducing or non-reducing conditions The isolated antibody was purified to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). includes antibodies in situ within recombinant cells, and at least one of the natural environments of the antibodies. In some cases, an isolated antibody is one that is free of at least one component. It is prepared by one purification step.

[0030] As used herein, the term "cytotoxic agent" refers to an agent that inhibits or agonizes the function of cells. refers to a substance that prevents and / or causes cell death or destruction. Also known as an "anti-tumor agent" The term "chemotherapeutic agents" refers to drugs used to treat cancer or other diseases or disorders. It can be a cytotoxic agent.

[0031] As used herein, the terms "treatment," "treating," and the like refer to the administration of a desired drug. The term refers to obtaining a physical and / or physiological effect. The effect may be complete or partial relief of a disease or its symptoms. may be prophylactic in that it prevents or partially prevents and / or reduces the severity of a disease and / or disorder It may be therapeutic in that it partially or completely cures the resulting side effects. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including a human. and (a) detecting a disease in a subject who is susceptible to the disease but has not been diagnosed with it. (b) inhibiting the disease, i.e., arresting its development and (c) to be disease-relieving, i.e., to cause regression of the disease. include.

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

[0033] A "therapeutically effective amount" or "effective amount" refers to a dose administered to a mammal or other subject to treat a disease. an amount of target-specific antibody that, when administered, is sufficient to affect such treatment in a disease; The term "therapeutically effective amount" refers to the amount of antibody, the disease and its severity, and the age and physical condition of the subject to be treated. Varies depending on weight etc.

[0034] As used herein, the term "refractory" 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. Refractory cancers may be resistant at the start of treatment or may become resistant during treatment. Refractory cancers may also be called resistant cancers.

[0035] "Biological sample" encompasses a variety of sample types obtained from an individual, including those used for diagnostic or monitoring purposes. It can be used in assays. The definition is blood and other liquid samples of biological origin, biopsy specimens solid tissue samples, such as tissue cultures or cells derived therefrom and their progeny. The definition also includes any modification of the protein after its acquisition in any manner, such as treatment with a reagent, solubilization, or samples that have been manipulated by enrichment for specific components, such as polynucleotides. The term "biological sample" encompasses clinical samples, including cells in culture, cell supernatants, cell lysates, blood Also included are serum, plasma, biological fluid, and tissue samples.

[0036] The percent identity between a pair of sequences is calculated by multiplying the number of matches in the pair by 100, including gaps. The identity score can be calculated by dividing the length of the aligned region by the length of the complete sequence. Only matches with similar amino acids are counted, regardless of how similar the amino acids are to each other. Internal gaps only is included in the length and does not include gaps at the ends of the sequence. Percent identity = (number of matches x 100 ) / length of aligned region (with gaps)

[0037] The phrase "conservative amino acid substitution" refers to substitution 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 replace amino acids in proteins with amino acids of similar side chains. By replacing the amino acid with an amino acid having a side chain of similar acidity, basicity, charge, polarity, or size, This can preserve the activity of the protein.

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

[0039] The term "vector" is used to transfer protein coding information to a host cell. any molecule or entity (e.g., a nucleic acid, a plasmid, a bacteriophage, or a virus) means.

[0040] The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell. , and (in conjunction with a host cell) expression of one or more heterologous coding regions operably linked thereto. An expression construct refers to a vector containing nucleic acid sequences that direct and / or control transcription, translation, and transcription. A gene that affects or controls the intron, if present, and is operably linked to it. These sequences may include, but are not limited to, sequences that affect RNA splicing of the coding region. It will not be done.

[0041] The term "stimulation" refers to the interaction of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) with Refers to the primary response induced by binding to the cognate ligand (or tumor antigen in the case of CARs) and thereby inhibiting, but not limited to, signaling through the TCR / CD3 complex; or Signaling pathways, such as signaling via the appropriate NK receptor or CAR signaling domains, are also important. Mediates signal transduction events. Stimuli can mediate altered expression of specific molecules. .

[0042] The term "stimulatory molecule" refers to a molecule that acts on at least some aspect of an immune cell signaling pathway. Immune cell signaling pathways that provide cytoplasmic signal sequences that regulate immune cell activation in a stimulatory manner. In one embodiment, the term "antigen" refers to a molecule expressed by a cell (e.g., a T cell, a NK cell, a B cell). In this study, the signal is transmitted between, for example, a peptide-loaded MHC molecule in the TCR / CD3 complex. These are primary signals initiated by the binding of Primary cytoplasmic synthases that act in a stimulatory manner, including but not limited to, mediating T cell responses. Signal transduction sequences (also called "primary signaling domains") are involved in the regulation of immunoreceptor activation. The present invention also provides signal transduction motifs known as ITAMs or synthase motifs. Examples of ITAM-containing cytoplasmic signaling sequences that are of particular use in FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, C including, but not limited to, those derived from D79b, DAP10, and DAP12. do not have.

[0043] The term "costimulatory molecule" refers to a molecule that specifically binds to a costimulatory ligand, thereby activating T cells. Co-stimulatory responses by T cells, including but not limited to, cognate binding proteins on T cells that mediate proliferation Costimulatory molecules refer to antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include MHC class I molecules, BTLA, and Toll ligand receptor, as well as OX40, CD27, CD28, CDS, and ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (C D137).

[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 is the immunotransducer of the CAR-containing cell, e.g., the CAR-T cell. For example, in CAR-T cells, Examples of vector functions include helper activity, including cytolytic activity and cytokine secretion. can be.

[0046] As used herein, an "immune effector cell" refers to a cell that mediates an immune response, e.g., an immune effector cell. These refer to cells that are involved in promoting immune effector responses. Examples of immune effector cells include T cells, For example, alpha / beta T cells and gamma / delta T cells, B cells, and natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes Included. 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 for administering the invention are also provided. the presence or absence of ABCC1 expression in cells, e.g., tumor cells; Using antibodies to detect levels and / or inhibit ABCC1 function Also disclosed are methods for administering to a subject an anti-ABCC1 antibody as disclosed herein. Also provided is a method for treating a subject for cancer, comprising:

[0048] Before describing the present invention in more detail, it is to be understood that the present invention is not limited to the particular embodiments described. As such, it should be understood that variations may occur. The terminology is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood that the scope of the present invention is limited only by the appended claims.

[0049] Where a range of values ​​is provided, the upper and lower limits of that range are and each intervening value to the tenth of the unit of the lower limit between and any other stated value within the stated range. It is understood that all narrower ranges, including any intervening values, are encompassed within the invention. The upper and lower limits may independently be included in the narrower ranges and any limit within the stated range may be included. The scope of the invention is encompassed within the scope of the invention, subject to the specific excluded limitations. Where a range includes one or both of the limits, a range excluding either or both of those limits is also included. Included in the present invention.

[0050] Certain ranges are provided herein by numerical values ​​preceded by the term "about." The term "about" is used herein to refer to the exact number that it precedes, as well as to its use. Provide textual support for numbers whose words are close to or approximately near the preceding number. Used to indicate whether a number is close to or approximately in the vicinity of a specifically listed number. In determining whether a non-listed number is close or approximately close, It may be any number that, in context, provides substantial equivalent value to the specifically recited number.

[0051] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present invention. The terms "a", "b", "c", "d" and "e" have the same meaning as commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. Although various methods and materials can be used, representative exemplary methods and materials are described here.

[0052] All publications and patents cited herein are to be construed as though each individual publication or patent were incorporated by reference. No. 6,037,793, filed Dec. 1, 2002, is incorporated herein by reference as if specifically and individually indicated to be incorporated herein by reference. The reference is made to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of any publication is hereby incorporated by reference for its disclosure prior to the filing date of this application. and the disclosure of any such material is hereby construed as an admission that the present invention is not entitled to antedate such publication. Furthermore, the dates of publication provided may differ from the actual publication dates, These may need to be independently verified.

[0053] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. Please note that "the" includes plural referents unless the context clearly indicates otherwise. It is further understood that the claims may be drafted to exclude any optional element. Therefore, this statement should not be construed as a "mere" reference to a recitation of claimed elements. Use of such exclusive terminology, such as "," "only," or the use of a "negative" limitation It is intended to serve as a reference.

[0054] As will be apparent to those skilled in the art upon reading this disclosure, the individual embodiments described and exemplified herein Each of the embodiments may be combined with several other embodiments without departing from the scope or spirit of the invention. individual components that can be easily separated from or combined with any of the features of Any enumerated method has the elements and properties of a recited sequence of events or a logically possible sequence of events. The steps may be performed in any other order possible.

[0055] The methods and compositions are described or explained for grammatical fluidity with functional explanations. However, claims must be expressly formulated under 35 U.S.C. 112(f). Unless otherwise specified, the invention is not necessarily construed as limited by the construction of "means" or "steps" limitations. Although the invention should not be limited to the definition provided by the claims under the judicial doctrine of equivalents, 112(f) of the U.S. Patent Act, giving the full scope of meaning and equivalents thereof. If expressly formulated under 35 U.S.C. § 112(f), full statutory equivalents under 35 U.S.C. It should be clearly understood that

[0056] antibody As summarized above, the present disclosure relates to a method for detecting and / or treating a mammalian cell, such as a human cell, that is expressed on the surface of the mammalian cell. The present invention provides an antibody that binds to the cellular efflux pump ABCC1, which is involved in glutathione-S-conjugation. as the ATPase ABCC1, or multidrug resistance-associated protein 1 (MRP1) ABCC1, also known as ABCC1, is an energy transporter that exports drugs and organic anions across the plasma membrane. It is a ATP-dependent pump. It contains 17 extracellular and cytoplasmic loops connected by ATP. ABCC1 is a cytotoxic molecule that inhibits the growth of mitochondrial cells, including the transmembrane helices. ... and mediates resistance to vincristine.

[0057] In some embodiments, the antibodies disclosed herein bind to the extracellular domain of ABCC1. In certain embodiments, the anti-ABCC1 antibodies of the present disclosure bind to one or more of the above sites. , binds to human ABCC1. In certain embodiments, the anti-ABCC1 antibodies of the present disclosure It binds to human ABCC1 expressed on the cell surface of human cells, for example, 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 chemotherapeutic agents, thereby reduce the IC50 of the therapeutic agent by at least 2-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) binds to cancer cells that express ABCC1 at higher levels than non-cancer cells , to a lower extent against ABCC1, resulting in significantly less binding to non-cancer cells. Has affinity.

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

[0060] The IC50 of a test antibody can be determined by measuring inhibition of cell growth. 0 is the antibody that produced half the maximal response, as determined by using the test antibody alone. The IC50 of the chemotherapeutic agent can be determined in the absence and presence of the test antibody. The IC50 of the antibody can be measured to determine the effect of the antibody on the chemotherapeutic agent. The cells may be cancer cells, such as daunorubicin, etoposide, vincristine, etc. The cancer cell line may be a doxorubicin-selected C1-positive variant of the lung cancer cell line, H69. The antibody may be H69AR, which is an antibody. When determining the IC50 of an antibody, the cells are The cells may be contacted with the antibody and the chemotherapeutic agent, and the antibody is tested in serial dilutions. The effect of the antibody on the IC50 of the drug can be determined by exposing the drug to a serial dilution of The cells are incubated at 37°C for a period of time (e.g., 24 to 84 hours). Cell viability was assessed using standard reagents and methods. The antibodies produced increase the sensitivity of cancer cells to treatment with chemotherapeutic agents, thereby The IC50 of a chemotherapeutic agent may be reduced by at least 5-fold. The antibody has an IC50 of the chemotherapeutic agent that is 5-fold or more, e.g., 6-fold or more, 7-fold or more, or 8-fold or more. , 9-fold or more, or 10-fold or more, for example, 5-10-fold.

[0061] In certain embodiments, one or more of the anti-ABCC1 antibodies disclosed herein This property is consistent with the ability of ABCC1 in animal models. It can be used to determine the safety of an antibody.

[0062] In certain embodiments, the anti-ABCC1 antibodies disclosed herein are specific for ABCC1. It is heterologous and does not show significant binding to other antigens.

[0063] In some embodiments, one or more of the subject antibodies is directed against cells expressing ABCC1. When bound to the cytoplasm, it can interfere with the function of the cellular ABCC1 protein. One or more of the antibodies may inhibit excretion by the ABCC1 protein, e.g., The efflux is, for example, 10% or more, 15% or more, compared to the efflux by ABCC1 in its absence. % or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, This includes cases where the level is reduced by 70% or more, 80% or more, or 90% or more, or 5% or more. In some embodiments, the subject antibodies exhibit a cytotoxic activity when bound to cells expressing ABCC1. or by other mechanisms, e.g., by leaking ABCC1. may interfere with the action of CC1, thereby increasing chemotherapeutic agent uptake and / or cell The survival rate of the patient may be reduced.

[0064] In certain embodiments, the variable heavy (VH) regions of the antibodies listed in Table 2 are and variable light chain (VL) region paired heavy chain complementarity determining regions (HCDRs) and light chain CDRs (LCDs) R) for binding to ABCC1. For example, in one embodiment, the anti-ABCC1 antibody of the present disclosure is an anti-ABCC1 antibody selected from the group consisting of C1 antibodies listed in Table 2. In certain embodiments, HCD competes with antibody .309 for binding to ABCC1. R1 to 3 and LCDR1 to 3 are defined according to the Kabat nomenclature.

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

[0066] To determine whether a first antibody competes with a second antibody for binding to ABCC1, Any suitable approach for detecting the first antibody may be used. Whether an antibody "competes with" an antibody can be determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, via 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 one of the two antibodies is added. One antibody is labeled. The labeled and unlabeled antibodies bind to separate and distinct sites on the antigen. In this case, the labeled antibody binds to the same level regardless of whether a suspected competing antibody is present or not. However, if the interaction sites are identical or overlapping, the unlabeled antibody will not compete. If unlabeled antibody is present in excess, the amount of labeled antibody bound to the antigen will decrease. In this case, very little, if any, labeled antibody will bind.

[0067] For purposes of this disclosure, a competing antibody inhibits the binding of an antibody to an antigen by about 30% or more, and preferably by about 40% or more. 0% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 85% or more, The antibody reduces the level of IL-1 by about 90% or more, about 95% or more, or about 99% or more. Detailed procedures for carrying out the method are well known in the art and are described, for example, in Harvard Medical School, low and Lane,Antibodies,A Laboratory Man ual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York,1988,567-569 , 1988, ISBN 0-87969-314-2. The assay can be quantitatively performed by using purified antibodies. can be established by titrating one antibody against itself, i.e., The antibody is used as both the label and the competitor. Binding of unlabeled competitor antibody to the antigen of the labeled antibody The ability to inhibit binding can be titrated. The results can be plotted and the amount of binding that is determined to achieve the desired degree of binding inhibition can be determined. The concentrations required for

[0068] In certain embodiments, the antibody that specifically binds to ABCC1 is (i) any of the antibodies listed in Table 2. The HCDRs 1 to 3 and HCDRs 4 of the pair of variable heavy chain (VH) and variable light chain (VL) regions of the antibody to be used are (ii) HCDRs 1 to 3 of the VH region of an antibody listed in Table 2 3, (iii) LCDR1 to LCDR3 of the VH region of an antibody listed in Table 2, or (iv) LCDR1 to LCDR3 of the VH region of an antibody listed in Table 2 HCDRs 1 to 3 of the VH region of the first antibody listed in Table 2 and HCDRs 1 to 3 of the VH region of the second antibody listed in Table 2 The VL region includes LCDR1 to LCDR3. HCDR and LCDR are designated by the Kabat nomenclature. It can be defined.

[0069] In certain embodiments, antibodies of the disclosure that specifically bind to human ABCC1 are those listed in Table 2. The HCDR1, HCDR2, and HCDR3 sequences of the listed antibodies, as well as LCDR1, L In addition to binding to human ABCC1, the present invention also provides a method for the production of ABCC1-specific antibodies, including CDR2, CDR3, and LCDR3 sequences. One or more of the antibodies provided may be from other mammalian species, such as mouse, monkey, or chimpanzee. The antibodies can be raised in mice or rats. The animals in which the antibodies were produced are shown. [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 are also referred to as anti-KPC1 antibodies or anti-C1 antibodies. These antibodies may be referred to by the antibody numbers listed in Table 2.

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

[0072] The antibodies of the present disclosure may be Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scF The antibody may be selected from the group consisting of: v, scAb, dAb, and Fv.

[0073] In some embodiments, the subject antibody is a recombinant or modified antibody, e.g., a chimeric antibody. As used herein, an antibody is an antibody that is humanized, deimmunized, or in vitro generated. In this case, the term "recombinant" or "modified" antibody refers to an antibody that has been (i) transfected into a host cell; (ii) antibodies expressed using recombinant recombinant expression vectors; (iii) antibodies isolated from antibody libraries, and (iv) human immunoglobulin gene-transduced antibodies. (iv) an antibody isolated from an animal (e.g., a mouse) into which the antibody was introduced; or (iv) a human immunoglobulin. prepared or expressed by any other means involving splicing of the gene sequence to other DNA sequences. prepared, expressed, produced, or isolated by recombinant means, such as antibodies It is intended to include all antibodies that are isolated. Such recombinant antibodies include those that are humanized, CDR modified, or modified. These include grafted, chimeric, deimmunized, and in vivo generated antibodies, optionally in vivo. It can include a constant region derived from a germline immunoglobulin sequence.

[0074] As described above, the subject anti-ABCC1 antibodies target one or more epitopes of ABCC1. It specifically binds to the ABCC1 epitope. The size of the ABCC1 epitope bound by the C1 antibody can vary, and the ABCC1 epitope For example, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11a a, 12aa, 4aa~10aa, 5aa~10aa, 6aa~10aa, 4aa~8a 3aa or less, including but not limited to a, 5aa-8aa, 6aa-8aa, etc. by polypeptides having continuous stretches of ABCC1 sequence that can range from 12 aa or more. This includes cases where the material is formed by

[0075] In some embodiments, the ABCC1 epitope is, for example, the human ABCC1 sequence :MALRGFCSADGSDPLWDWNVTWNTSNPDFTKCFQNTVLV WVPCFYLWACFPFYFLYLSRHDRGYIQMTPLNKTKTALGF LLWIVCWADLFSFWERSRGIFLAPVFLVSPTLLGITMLL ATFLIQLERRKGVQSSGIMLTFWLVALVCALAIRSKIMT ALKEDAQVDLFRDITFYVYFSLLLIQLVLSCFSDRSPLFS ETIHDPNPCPESSAS FLSRITFWWITGLIVRGYRQPLEGS DLWSLNKEDTSEQVVPVLVKNWKKECAKTRKQPVKVVYSS KDPAQPKESSKVDANEEVEALIVKSPQKEWNPSLFKVLYK TFGPYFLMSFFFKAIHDLMMFSGPQILKLLIKFVNDTKAP DWQGYFYTVLLFVTACLQTLVLHQYFHICFVSGMRIKTAV IGAVYRKALVITNSARKSSTVGEIVNLMSVDAQRFMDLAT YINMIWSAPLQVILALYLLWLNLGPSVLAGVAVMVLMPVPV NAVMAMKTKTYQVAHMKSKDNRIKLMNEILNGIKVLKLYA WELAFKDKVLAIRQEELKVLKKSAYLSAVGTFTWVCTPFL VALCTFAVYVTIDENNILDAQTAFVSLALFNILRFPLNIL PMVISSIVQASVSLKRLRIFLSHEELEPDSIERRPVKDGG GTNSITVRNATFTWARSDPPTLNGITFSIPEGALVAVVGQ VGCGKSSLLSALLAEMDKVEGHVAIKGSVAYVPQQAWIQN DSLRENILFGCQLEEPYYRSVIQACALLPDLEILPSGDRT EIGEKGVNLSGGQKQRVSLARAVYSNADIYLFDDPLSAVD AHVGKHIFENVIGPKGMLKNKTRILVTHSMSYLPQVDVII VMSGGKISEMGSYQELLARDGAFAEFLRTYASTEQEQDAE ENGVTGVSGPGKEAKQMENGMLVTDSAGKQLQRQLSSSSS YSGDISRHHNSTAELQKAEAKKEETWKLMEADKAQTGQVK LSVYWDYMKAIGLFISFLSIFLFMCNHVSALASNYWLSLW TDDPIVNGTQEHTKVRLSVYGALGISQGIAVFGYSMAVSI GGILASRCLHVDLLHSILRSPMSFFERTPSGNLVNRFSKE LDTVDSMIPEVIKMFMGSLFNVIGACIVILLATPIAAIII PPLGLIYFFVQRFYVASSRQLKRLESVSRSPVYSHFNETL LGVSVIRAFEEQERFIHQSDLKVDENQKAYYPSIVANRWL AVRLECVGNCIVLFAALFAVISRHSLSAGLVGLSVSYSLQ VTTYLNWLVRMSSEMETNIVAVERLKEYSETEKEAPWQIQ ETAPPSSWPQVGRVEFRNYCLRYREDLDFVLRHINVTING GEKVGIVGRTGAGKSSLTLGLFRINESAEGEIIIDGINIA KIGLHDLRFKITIIPQDPVLFSGSLRMNLDPFSQYSDEEV WTSLELAHLKDFVSALPDKLDHECAEGGENLSVGQRQLVC LARALLRKTKILVLDEATAAVDLETDDLIQSTIRTQFEDC TVLTIAHRLNTIMDYTRVIVLDKGEIQEYGAPSDLLQQRG LFYSMAKDAGLV (SEQ ID NO: 150), or an extracellular domain thereof, e.g., Pair 1 (amino acids 1-33): MALRGFCSADGSDPLWDWNVTWNTSNP DFTKCF (SEQ ID NO: 151), Loop 2 (amino acids 96-100): RSRGI (SEQ ID NO: 152), Sequence number 152), loop 3 (amino acids 155-172): SKIMTALKEDAQVD LFRD (SEQ ID NO: 153), loop 4 (amino acids 338 to 363): MMFSGPQI LKLLIKFVNDTKAPDWQG (SEQ ID NO: 154), loop 5 (amino acids 462 ~464): LGP, Loop 6 (amino acids 569-690): VTIDENNILDAQ TAFVSLALFN (SEQ ID NO: 155), loop 7 (amino acids 989 to 1025): A LASNYWLSLWTDDPIVNGTQEHTKVRLSVYGALGIS(Sequence number No. 156), loop 8 (amino acids 1111): A, loop 9 (amino acids 1225-1222): B 6): LS, or a fragment of human ABCC1, for example, containing amino acid residues 204 to 1531 Fragment:MDPNPCPESSASFLSRITFWWITGLIVRGYRQPLEGS DLWSLNKEDTSEQVVPVLVKNWKKECAKTRKQPVKVVYSS KDPAQPKESSKVDANEEVEALIVKSPQKEWNPSLFKVLYK TFGPYFLMSFFFKAIHDLMMFSGPQILKLLIKFVNDTKAP DWQGYFYTVLLFVTACLQTLVLHQYFHICFVSGMRIKTAV IGAVYRKALVITNSARKSSTVGEIVNLMSVDAQRFMDLAT YINMIWSAPLQVILALYLLWLNLGPSVLAGVAVMVLMVPV NAVMAMKTKTYQVAHMKSKDNRIKLMNEILNGIKVLKLYA WELAFKDKVLAIRQEELKVLKKSAYLSAVGTFTWVCTPFL VALCTFAVYVTIDENNILDAQTAFVSLALFNILRFPLNIL PMVISSIVQASVSLKRLRIFLSHEELEPDSIERRPVKDGG GTNSITVRNATFTWARSDPPTLNGITFSIPEGALVAVVGQ VGCGKSSLLSALLAEMDKVEGHVAIKGSVAYVPQQAWIQN DSLRENILFGCQLEEPYYRSVIQACALLPDLEILPSGDRT EIGEKGVNLSGGQKQRVSLARAVYSNADIYLFDDPLSAVD AHVGKHIFENVIGPKGMLKNKTRILVTHSMSYLPQVDVII VMSGGKISEMGSYQELLARDGAFAEFLRTYASTEQEQDAE ENGVTGVSGPGKEAKQMENGMLVTDSAGKQLQRQLSSSSS YSGDISRHHNSTAELQKAEAKKEETWKLMEADKAQTGQVK LSVYWDYMKAIGLFISFLSIFLFMCNHVSALASNYWLSLW TDDPIVNGTQEHTKVRLSVYGALGISQGIAVFGYSMAVSI GGILASRCLHVDLLHSILRSPMSFFERTPSGNLVNRFSKE LDTVDSMIPEVIKMFMGSLFNVIGACIVILLATPIAAIII PPLGLIYFFVQRFYVASSRQLKRLESVSRSPVYSHFNETL LGVSVIRAFEEQERFIHQSDLKVDENQKAYYPSIVANRWL AVRLECVGNCIVLFAALFAVISRHSLSAGLVGLSVSYSLQ VTTYLNWLVRMSSEMETNIVAVERLKEYSETEKEAPWQIQ ETAPPSSWPQVGRVEFRNYCLRYREDLDFVLRHINVTING GEKVGIVGRTGAGKSSLTLGLFRINESAEGEIIIDGINIA KIGLHDLRFKITIIPQDPVLFSGSLRMNLDPFSQYSDEEV WTSLELAHLKDFVSALPDKLDHECAEGGENLSVGQRQLVC LARALLRKTKILVLDEATAAVDLETDDLIQSTIRTQFEDC TVLTIAHRLNTIMDYTRVIVLDKGEIQEYGAPSDLLQQRG LFYSMAKDAGLV (SEQ ID NO: 157), or the cynomolgus monkey ABCC1 sequence: MALRGFCSADGSDPLWDWNVTWYTSNPDFTKCFQNTVLVW VPCFYLWACFPFYFLYLSRHDRGYIQMTLLNKTKTALGFL LWIVCWADLFYSFWERSRGIFLAPVFLVSPTLLGITMLLA TFLIQLERRKGVQSSGIMLTFWLVALLCALAILRSKIMTA LKEDVQVDLFRDMTFYVYFSLVLIQLVLSCFSDRSPLFSE TIHDPNPCPESSASFLSRITFWWITGLIVRGYRQPLEGSD LWSLNKEDTSEQVVPVLVKNWKKECAKTRKQPVKVYSSK DPAQPKDSSKVDANEEVEALIVKSPQKEWNPSLFKVLYKT FGPYFLMSFFFKAIHDLMMFSGPEIKLLINFVNDTKAPD WQGYFYTALLFVAACLQTLVLHQYFHICFVSGMRIKTAVI GAVYRKALVITNAARKSSTVGEIVNLMSVDAQRFMDLATY INMIWSAPLQVILALYLLWRNLGPPILAGVAVMVLMVPVN AVMAMKTKTYQVAHMKSKDNRIKLMNEILNGIKVLKLYAW ELAFKDKVLAIRQEELKVLKKSAYLAAVGTFTWVCTPFLV ALCTFAVYVTIDKNNVLDAQKAFVSLALFNILRFPLNILP MVISSIVQASVSLKRLRIFLSHEELEPDSIERRPVKDGGD TNSITVRNATFTWARSDPPTLNGITFSIPEGALVAVVGQV GCGKSSLLSALLAEMDKVEGHVALKGSVAYVPQQAWIQND SLQENILFGCQLEEPYYRSVIQACALLPDLEILPSGDRTE IGEKGVNLSGGQKQRVSLARAVYCNADIYLFDDPLSAVDA HVGKHIFENVIGPKGMLKNKTRILVTHSMSYLPQVDVIIV MSGGKISEMGSYQELLARDGAFAEFLRTYASAEQEQDPED NGVTGVSGPGKEAKQMENGMLVTDSAGKQLQRQLSSSSSSY SGDVSRQHNSTAELQKDGAKKEETWKLMEADKAQTGQVKL SVYWDYMKAIGLFISFLSILFICNHVAALASNYWLSLWT DDPIVNGTQEHTKVRLSVYGALGISQGIAVFGYSMAVSIG GILASRCLHVDLLHSILRSPMSFFERTPSGNLVNRFSKEL DTVDSMIPEVIKMFMGSLFNVIGACIVILLATPIAAIIIP PLGLIYFFVQRFYVASSRQLKRLESVSRSPVYSHFNETLL GVSVIRAFEEQERFIHQSDLKVDENQKAYYPSIVANRWLA VRLECVGNCIVLFAALFAVISRHSLSAGLVGLSVSYSLQV TTYLNWLVRMSSEMETNIVAVERLKEYSETEKEAPWQIQE TAPPSNWPQVGRVEFRNYCLRYREDLDFVLRHINVTINGG EKVGIVGRTGAGKSSLTLGLFRINESAEGEIIIDGINIAR IGLHDLRFKITIIPQDPVLFSGSLRMNLDPFSQYSDEEVW TSLELAHLKGFVSALPDKLDHECAEGGENLSVGQRQLVCL ARALLRKTKILVLDEATAAVDLETDDLIQSTIRTQFEDCT VLTIAHRLNTIMDYTRVIVLDKGEIQEYGAPSDLLQQRGL FYNMARDAGLV (SEQ ID NO: 158), its extracellular domain, or cynomolgus ABC A fragment of C1, for example, a fragment comprising amino acid residues 204 to 1531: MDPNPCPESSASFLSRITFWWITGLIVRGYRQPLEGSDL WSLNKEDTSEQVVPVLVKNWKKECAKTRKQPVKVVYSSKD PAQPKDSSKVDANEEVEALIVKSPQKEWNPSLFKVLYKTF GPYFLMSFFFKAIHDLMMFSGPEILKLLINFVNDTKAPDW QGYFYTALLFVAACLQTLVLHQYFHICFVSGMRIKTAVIG AVYRKALVITNAARKSSTVGEIVNLMSVDAQRFMDLATYI NMIWSAPLQVILALYLLWRNLGPPILAGVAVMVLMVPVNA VMAMKTKTYQVAHMKSKDNRIKLMNEILNGIKVLKLYAWE LAFKDKVLAIRQEELKVLKKSAYLAAVGTFTWVCTPFLVA LCTFAVYVTIDKNNVLDAQKAFVSLALFNILRFPLNILPM VISSIVQASVSLKRLRIFLSHEELEPDSIERRPVKDGGDT NSITVRNATFTWARSDPPTLNGITFSIPEGALVAVVGQVG CGKSSLLSALLAEMDKVEGHVALKGSVAYVPQQAWIQNDS LQENILFGCQLEEPYYRSVIQACALLPDLEILPSGDRTEI GEKGVNLSGGQKQRVSLARAVYCNADIYLFDDPLSAVDAH VGKHIFENVIGPKGMLKNKTRILVTHSMSYLPQVDVIIVM SGGKISEMGSYQELLARDGAFAEFLRTYASAEQEQDPEDN GVTGVSGPGKEAKQMENGMLVTDSAGKQLQRQLSSSSSYS GDVSRQHNSTAELQKDGAKKEETWKLMEADKAQTGQVKLS VYWDYMKAIGLFISFLSIFLFICNHVAALASNYWLSLWTD DPIVNGTQEHTKVRLSVYGALGISQGIAVFGYSMAVSIGG ILASRCLHVDLLHSILRSPMSFFERTPSGNLVNRFSKELD TVDSMIPEVIKMFMGSLFNVIGACIVILLATPIAAIIIPP LGLIYFFVQRFYVASSRQLKRLESVSRSPVYSHFNETLLG VSVIRAFEEQERFIHQSDLKVDENQKAYYPSIVANRWLAV RLECVGNCIVLFAALFAVISRHSLSAGLVGLSVSYSLQVT TYLNWLVRMSSEMETNIVAVERLKEYSETEKEAPWQIQET APPSNWPQVGRVEFRNYCLRYREDLDFVLRHINVTINGGE KVGIVGRTGAGKSSLTLGLFRINESAEGEIIIDGINIARI GLHDLRFKITIIPQDPVLFSGSLRMNLDPFSQYSDEEVWT SLELAHLKGFVSALPDKLDHECAEGGENLSVGQRQLVCLA RALLRKTKILVLDEATAAVDLETDDLIQSTIRTQFEDCTV LTIAHRLNTIMDYTRVIVLDKGEIQEYGAPSDLLQQRGLF ABCC1, including but not limited to YNMARDAGLV (SEQ ID NO: 159) For continuous extension of the sequence, at least about 75%, at least about 80%, at least about 85% %, at least about 90%, at least about 95%, at least about 98%, at least about 99 %, or 100% amino acid sequence identity.

[0076] A subject anti-ABCC1 antibody exhibits high affinity binding to ABCC1. For example, a subject anti-ABCC1 antibody The BCC1 antibody binds to human ABCC1 with a specific 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, young Or at least about 10 -12 M or 10 -12 It binds with an affinity greater than M. The ABCC1 antibody targets an epitope present in ABCC1 with 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 With affinity exceeding M and combine.

[0077] The subject anti-ABCC1 antibodies may be used in combination with related but different sequence EPs, such as related but different sequence EPs. Binding to any epitope formed by amino acids in different proteins is essentially The amino acids formed by proteins that are related but differ in sequence are not shown. Any binding of a subject anti-ABCC1 antibody to a pitope will generally be consistent with the expression of an endogenous ATP in ABCC1. Nonspecific binding of anti-ABCC1 antibodies with substantially lower affinity than the specific binding of the anti-ABCC1 antibodies to the target cytoplasm. 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] The subject anti-ABCC1 antibodies are those that bind to the ABCC1 transporter, e.g., human ABCC1. For example, a subject anti-ABCC1 antibody can reduce transport of an anti-ABCC1 molecule. at least about 5%, at least about 10%, or at least about 5% of the transport in the absence of ABCC1 antibody. 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 can be reduced by 70%, at least about 80%, at least about 90%, or more .

[0079] In some embodiments, the subject antibodies are expressed in, for example, rodents, non-human primates, and humans. a mammalian gene encoding a heavy chain FR sequence (e.g., encoded by each heavy chain FR coding sequence) It contains the FR region, which is a sequence.

[0080] The subject antibodies are those having the sequences for the VH region of the VH-VL pair of the antibodies described in Table 2. ,including 100%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity The subject antibodies may comprise a heavy chain variable (VH) region comprising the amino acid sequence described in Table 2. For the sequence of the VL region of the VH-VL region pair of the antibody to be used, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 96%, 97%, 98%, or 99% or more identical amino acid sequence. It may contain a variable (VL) region.

[0081] In one embodiment, the antibody molecule is a C1.844 antibody, a C1.851 antibody listed in Table 2. HCDR1-3 and / or L of one of the C1.831 antibody, C1.861 antibody, Includes CDR1 to 3.

[0082] In one embodiment, the antibody molecule is a C1.773 antibody, a C1.773a antibody, or any of the antibodies listed in Table 2. body, C1.777a antibody, C1.784a antibody, C1.786a antibody, C1.787a anti body, C1.827 antibody, C1.830B antibody, C1.831 antibody, C1.835 antibody, C 1.841 antibody, C1.844 antibody, C1.845 antibody, C1.847 antibody, C1.85 1 antibody, C1.855 antibody C1.861 antibody, C1.863 antibody, C1.876 antibody, C HCDR1 to 3 and / or LC of one of the C1.877 antibody or C1.879A antibody Includes DR1~3.

[0083] In certain embodiments, the antibodies of the present disclosure inhibit the activity of chemotherapeutic agents (e.g., vincristine). IC50 is increased by 5 times or more, for example, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more. In one embodiment, the antibody molecule may be reduced by 20-fold or more, for example, by 5-50-fold. The IC50 of a chemotherapy agent (e.g., vincristine) can be reduced by more than five-fold, and are listed in Table 2. C1.851 antibody, C1.841 antibody, C1.861 antibody, C1.831 antibody, C1 HCDRs 1 to 3 and / or HCDRs 1 to 3 of the C1.786a antibody, the C1.787a antibody, or the C1.777 antibody may include LCDR1 to LCDR3.

[0084] The regions and / or chains of the subject antibodies may be joined or linked by one or more linker regions. If present, the linker region may be from about 5 amino acids to about 50 amino acids in length. amino acids, for example, having a length of about 5 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about Approx. 20aa, approx. 20aa ~ approx. 25aa, approx. 25aa ~ approx. 30aa, approx. 30aa ~ approx. 35a a, about 35 aa to about 40 aa, about 40 aa to about 45 aa, or about 45 aa to about 50 aa There can be.

[0085] Suitable linkers for use with the subject antibodies include "flexible linkers." When used in conjunction with a linker molecule, the linker molecule generally allows some degree of flexible movement between the linked regions. Linker molecules are generally about 6 to 50 atoms long. The linker molecule may be, for example, an arylacetylene, an ethylenediamine containing 2 to 10 monomer units, or may be a glycol oligomer, a diamine, a diacid, an amino acid, or a combination thereof. Other linker molecules capable of binding to polypeptides may be used in view of the present disclosure. It is possible.

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

[0087] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine Rimer (e.g., (GS) n , G.S.G.S.G.S. n (SEQ ID NO: 160), and GGGS n (array No. 161), wherein n is an integer of at least 1), glycine-alanine poly mers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers contain both of these amino acids. is of interest because it is relatively unstructured and can therefore act as a neutral tether between components. Glycine polymers are those in which glycine is more abundant than alanine in the pi-psi space. are significantly more accessible than residues with longer side chains and are much less restricted than residues with longer side chains. , which are of particular interest (Scheraga, Rev. Computational Chem. 11173-142 (1992). Linkers include GGSG (SEQ ID NO: 162), GGSGG (SEQ ID NO: 163), GSGS G (SEQ ID NO: 164), GSGGG (SEQ ID NO: 165), GGGSG (SEQ ID NO: 166) , GSSSG (SEQ ID NO: 167), and the like. The design of the peptide conjugated to any of the above elements may be wholly or partially flexible. The linker may include a linker that is flexible, thereby making the linker a flexible linker. and one or more portions that provide a less flexible structure. You will recognize it.

[0088] In other instances, flexibility in the hinge region of the antibodies of the present disclosure is enhanced by replacing amino acid C220 with serine. or any other natural amino acid to remove C220. Therefore, by removing the complete hinge or replacing the IgG1 hinge with an IgG3 hinge, It can be reduced by either replacing the IgA2 protein with the IgA2 protein, which is found in humans with the isotype IgA2m. Similar to the situation, antibodies are formed in which the light chains are connected via their C-terminal cysteines. This reduces the flexibility of the Fab relative to the Fc and consequently reduces its cross-linking ability. Another strategy to reduce the flexibility of the IgG1 molecule is to replace the IgG1 hinge with an IgG2 hinge or I Alternatively, an IgG1 hinge similar to the IgG2 hinge may be used. A variant of the hinge can be introduced. This mutant (TH7Δ6-9) contains additional To generate a shorter hinge with a cysteine, the mutation T223C and two deletions ( K222 and T225).

[0089] The replacement of mouse CDRs into the human variable domain framework ensures their correct spatial orientation For example, a human variable domain framework can result in the retention of CDRs. The murine variable domain framework adopts the same or similar conformation. The framework sequences are highly similar to the mouse variable framework domains from which the CDRs are derived. This can be achieved by obtaining human variable domains from human antibodies that share sequence identity. 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 sequences of naturally occurring human antibodies. , or a consensus sequence of several human antibodies. ough et al.,Protein Engineering 4:773(19 91), Kolbinger et al., Protein Engineering 6:971 (1993).

[0090] Complementarity determination of mouse donor immunoglobulins and appropriate human acceptor immunoglobulins The regions have been identified and the next step is to identify residues from these components, if any, The goal is to determine which residues to replace in order to optimize the properties of the resulting humanized antibody. In general, the introduction of murine residues induces a human anti-murine antibody (HAMA) response in humans. Substitution of human amino acid residues by mouse is minimized to prevent the risk of antibody reactions. Art-recognized methods for determining immune responses should be performed to determine the specific HAMA responses can be monitored in patients or during clinical trials. Patients receiving the therapy should undergo immunogenicity assessment at the start and throughout the course of the therapy. The HAMA response can be measured, for example, by surface plasmon resonance technology (BIACORE) and / or or in serum samples from patients using methods known to those skilled in the art, including solid-phase ELISA assays. and by detecting antibodies against the humanized therapeutic reagent. In this case, the subject humanized antibodies do not substantially induce a HAMA response in human subjects.

[0091] Certain amino acids from the human variable region framework residues are used to determine CDR conformation and / or Substitutions are selected based on their possible effect on binding to the antigen. The non-native juxtaposition of the CDR regions with the human variable framework regions results in conformational constraints. resulting in a decrease in binding affinity unless corrected by substitution of specific amino acid residues. .

[0092] The selection of amino acid residues for substitution is determined, in part, by computer modeling. A computer hardware system for generating three-dimensional images of immunoglobulin molecules can be used. Hardware and software are known in the art. Generally, molecular models are , are generated starting from the solved structures of immunoglobulin chains or domains thereof. The chains to be modeled are selected based on amino acid sequence similarity to chains or domains of solved three-dimensional structure. The chain or domain showing the greatest sequence similarity is the starting point for building the molecular model. Chains or domains that share at least 50% sequence identity are selected as models. Preferably, at least 60%, 70%, 80%, 90% of the sequences are selected for sequencing. Those that share identity or more are selected for modeling. The starting structure is a comparison of the actual amino acids in the immunoglobulin chain or domain being modeled with the starting structure. The modified structure is then modified to allow for differences between the amino acids in the structure. Finally, the model is analyzed by energy minimization. and all atoms are within appropriate distances from one another and bond lengths and angles are chemically permissible. It is refined by verifying that it is within the bounds of what is possible.

[0093] In some embodiments, the subject antibodies comprise scFv multimers. In some embodiments, the subject antibodies are scFv dimers (e.g., two tandem scFvs ( scFv trimers (e.g., three tandem scFvs (scFv3)), scFv trimers (e.g., three tandem scFvs (scFv3)), ), scFv tetramers (e.g., containing four tandem scFvs (scFv4)) or is a multimer of more than four scFvs (e.g., in tandem). The monomers may be linkers of about 2 amino acids to about 15 amino acids in length, e.g., 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11a linked in tandem via a linker of 1a, 12aa, 13aa, 14aa, or 15aa. Suitable linkers include, for example, (Gly)x (SEQ ID NO: 168) where x is an integer from 2 to 15. Other suitable linkers are those discussed above. In some embodiments, each of the scFv monomers in a subject scFv multimer In certain embodiments, the bispecific antibody is humanized as described in the literature. For example, the bispecific antibodies of the present disclosure may be in any molecular format known in the art. Spiess C.et al.,Mol Immunol.2015 Oct;67( 2 Pt A):95-106.

[0094] In some embodiments, the subject antibodies comprise a constant region of an immunoglobulin (e.g., Fc The Fc region, if present, can be a human Fc region. If present, the antibody can include both light and heavy chain constant regions. The constant region includes CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein are of all types, including IgM, IgG, IgD, IgA, and IgE. Common regions and any isotype, including IgG1, IgG2, IgG3, and IgG4 An example of a suitable heavy chain Fc region is an antibody of human isotype IgG1 Fc. The light chain constant region can be lambda or kappa. The humanized antibodies of the invention may contain sequences from more than one class or isotype. Antibodies are 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 a mixture of heavy and light chain variable domains. It can be expressed as a single chain antibody linked via a pacer.

[0095] In some embodiments, the subject antibodies have a free thiol (-SH) at the carboxyl terminus. ) group, and the free thiol group can be used to couple the antibody to a second polypeptide (e.g., an antibody of interest) The antibody may be attached to another antibody, including a scaffold, carrier, or the like.

[0096] The subject antibodies may be crosslinked using, for example, glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. A crosslinking agent is used to attach a second moiety (e.g., a lipid, a polypeptide other than the antibody of interest, a synthetic polypeptide, Glutaraldehyde can be used to covalently bind to polymers (polymers, carbohydrates, toxins, etc.). Peptides are cross-linked via their amino moieties. Monobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters ester, or homobifunctional sulfhydryl-reactive crosslinkers, contain two or more identical reactive moieties. Once the cross-linker is added to a solution containing the mixture of polypeptides to be linked, Homobifunctional NHS esters and imide esters can be used in the STEP reaction procedure. At mildly alkaline pH, imide esters cross-link amine-containing polypeptides. The ester reacts only with primary amines to form imideamides, which are essential for cross-linked polypeptides. The overall charge is unaffected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimides. Dohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB) , and 1,4-bis[3-(2-pyridyldithio)propionamido]butane (DPDP B) is included.

[0097] bispecific antibody In certain embodiments, the antibodies provided herein are those described in the preceding sections. and a bispecific antibody comprising the VH and VL regions of an anti-ABCC1 antibody such as a second VH region comprising HCDRs 1 to 3 of an antibody that binds to a tumor-associated antigen (TAA), include.

[0098] The antibody can include a second VL region, and the second VH region and the second VL region are linked to a TAA. In certain embodiments, the second VH region and the second VL region are combined into a single polypeptide. In certain embodiments, the second VH region and the second VL region may be present in a peptide. , present in the scFv.

[0099] In certain embodiments, the bispecific antibody comprises a common light chain, the common light chain being a light chain selected from the group consisting of nucleotides of the nucleotides described above. The VL region of an anti-ABCC1 antibody as described in the section.

[0100] The TAAs can be any antigen known to be overexpressed in cancer cells For example, TAAs are not expressed at detectable levels in normal cells but are expressed in cancer cells. The antigen is expressed by the normal and cancer cells, and the normal and cancer cells are of the same cell type, e.g., epithelial cells. For example, a TAA may convert the amino acid sequence of an encoded protein to the amino acid sequence of the unmutated protein. Neoantigens are a class of tumor antigens that arise from tumor-specific mutations that alter the amino acid sequence relative to the target antigen. In other embodiments, the TAA is expressed in normal cells but more abundantly in cancer cells. It is an antigen that is expressed at high levels.

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

[0102] In certain embodiments, the bispecific antibodies are C1 and C2 as listed in Table 2, respectively. VH region and VL region of antibody C1.844 or antibody C1.851, including HCDR1 to HCDR3. The H region and the VL region including LCDR1-3, and the HCDR1-3 of atezolizumab, respectively and a second VH region comprising LCDR1-3 and LCDR1-3, and a second VL region. In embodiments, HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature. will be done.

[0103] In certain embodiments, the bispecific antibody is a C1.844 antibody listed in Table 2 or The VH and VL regions of the C1.851 antibody and HCDR1-3 and LC of atezolizumab and an scFv comprising HCDR1 to 3 and LCDR1 to 3, wherein HCDR1 to 3 and LCDR1 to 3 are derived from Kaba It is defined according to the naming.

[0104] In certain embodiments, the bispecific antibodies are C1 and C2 as listed in Table 2, respectively. HCD of the VH and VL regions of the .844hu21 antibody or the C1.851hu12 antibody a VH region including R1 to R3 and a VL region including LCDR1 to LCDR3, and a second VH region and a second VL region comprising HCDRs 1 to 3 and LCDRs 1 to 3 of the first VH region; In certain embodiments, HCDRs 1-3 and LCDRs 1-3 may comprise the sequences of Kabat Defined according to naming.

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

[0106] In certain embodiments, present in the second VH region or scFv region of the bispecific antibody HCDR1 to HCDR3 are those of atezolizumab, and HCDR1 has the sequence D SWIH (SEQ ID NO: 25), and HCDR2 contains the sequence WISPYGGSTYYADSV KG (SEQ ID NO: 169), and HCDR3 contains the sequence RHWPGGFDY (SEQ ID NO: 17 In certain embodiments, bispecific antibodies that bind to ABCC1 and PD-L1 include The antibody may further comprise a second VL region comprising LCDRs 1-3 of atezolizumab, LCDR1 contains the sequence RASQDVSTAVA (SEQ ID NO: 171), and LCDR2 contains the sequence SA SFLYS (SEQ ID NO: 172), and LCDR3 contains the sequence QQYLYHPAT (SEQ ID NO: 173). No. 173). In a specific embodiment, LCDRs 1 to 3 present in the scFv region contains LCDR1-3 of atezolizumab, defined according to Kabat nomenclature.

[0107] In certain embodiments, the bispecific antibody that binds to ABCC1 and PD-L1 comprises: The amino acid sequence of the VH region of atezolizumab as set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQ APGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS( at least 80%, at least 90%, at least 95%, or contains a second VH region that contains an amino acid sequence of 100% identity.

[0108] In certain embodiments, the bispecific antibody that binds to ABCC1 and PD-L1 comprises: The amino acid sequence of the VL region of atezolizumab as set forth below: Diqmtqspsslsasvgdrvtitcrasqdvstavawyqqk pgkapklliysasflysgvpsrfsgsgsgtdftltisslq For pedfatyycqqylyhpatfgqgtkveik (SEQ ID NO: 175) at least 80%, at least 90%, at least 95%, or 100% identical to the The second VL region comprises the amino acid sequence

[0109] In certain embodiments, the bispecific antibody that binds to ABCC1 and PD-L1 is Binds to D-L1 and has the amino acid sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQ APGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS QDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEI At least 80%, at least 90%, at least 95% relative to K (SEQ ID NO: 176) or scFv regions comprising amino acid sequences of 100% identity.

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

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

[0112] In certain embodiments, the bispecific antibody is selected from the group consisting of C1.84, C2.85, C3.86, C4.87, C5.88, C6.89, C7.89, C8.89, C9.89, C10.89, C11.89, C12.89, C13.89, C14.89, C15.89, C16.89, C17.89, C1 HCDRs of the VH and VL regions of antibody C1.4, antibody C1.831, or antibody C1.851 a VH region containing LCDR1 to LCDR3 and a VL region containing LCDR1 to LCDR3, respectively; a second VH region and a second VL region comprising HCDRs 1 to 3 and LCDRs 1 to 3 of the In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are selected from the group consisting of HCDRs 1-3 according to the Kabat nomenclature. It is therefore defined.

[0113] In certain embodiments, the bispecific antibody is selected from the group consisting of C1.84, C2.85, C3.86, C4.87, C5.88, C6.89, C7.89, C8.89, C9.89, C10.89, C11.89, C12.89, C13.89, C14.89, C15.89, C16.89, C17.89, C1 HCDRs of the VH and VL regions of antibody C1.4, antibody C1.831, or antibody C1.851 a VH region including LCDRs 1 to 3 and a VL region including LCDRs 1 to 3, and the HCDR of trastuzumab and an scFv comprising LCDRs 1 to 3 and LCDRs 1 to 3.

[0114] In certain embodiments, the bispecific antibody is a C1.844hu21 antibody, a C1.83 The VH and VL regions of the C1.851hu11 antibody or the C1.851hu12 antibody and Trastuzumab and an scFv comprising HCDRs 1-3 and LCDRs 1-3 of a mab, and LCDR1 to LCDR3 are defined according to Kabat nomenclature.

[0115] In certain embodiments, present in the second VH region or scFv region of the bispecific antibody The corresponding HCDRs 1 to 3 are those of trastuzumab, and HCDR1 has the sequence D TYIH (SEQ ID NO: 177), and HCDR2 contains the sequence RIYPTNGYTRYADS VKG (SEQ ID NO: 178), and HCDR3 contains the sequence WGGDGFYAMDY (SEQ ID NO: In certain embodiments, dual specific antibodies that bind to ABCC1 and HER2 are included. The heteromeric antibody may further comprise a second VL region comprising LCDR1-3 of trastuzumab, DR1 contains the sequence RASQDVNTAVA (SEQ ID NO: 180) and LCDR2 contains the sequence SASFLYS (SEQ ID NO: 172), and LCDR3 contains the sequence QQHYTTPPT (sequence In certain embodiments, the LCDR1 present in the scFv region comprises: ~3 contains LCDR1~3 of trastuzumab, defined according to Kabat nomenclature.

[0116] In certain embodiments, the bispecific antibody that binds to ABCC1 and HER2 comprises: The amino acid sequence of the VH region of trastuzumab as described in: Evqlvesggglvqpggslrlscaasgfnikdtyihwvrq apgkglewvariyptngytryadsvkgrftisadtsknta ylqmnslraedtavyycsrwggdgfyamdywgqgtlvtvs At least 80%, at least 90%, at least 95% relative to s (SEQ ID NO: 182) or a second VH region comprising an amino acid sequence of 100% identity.

[0117] In certain embodiments, the bispecific antibody that binds to ABCC1 and HER2 comprises: The amino acid sequence of the VL region of trastuzumab as described in: Diqmtqspsslsasvgdrvtitcrasqdvntavawyqqk pgkapklliysasflysgvpsrfsgsrsgtdftltisslq pedfatyycqqhyttpptfgqgtkveik (SEQ ID NO: 183) at least 80%, at least 90%, at least 95%, or 100% identical to the The second VL region comprises the amino acid sequence

[0118] In certain embodiments, the bispecific antibody that binds to ABCC1 and HER2 is Binds to R2 and has the amino acid sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQ APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGCGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCR ASQDVNTAVAWYQQKPGKCPKLLIYSASFLYSGVPSRFSG SRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKV At least 80%, at least 90%, at least 9% The scFv region contains an amino acid sequence that is 5% or 100% identical.

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

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

[0121] The heavy chain constant region and the light chain constant region are those of a human IgG antibody, for example, a human IgG1 antibody. The constant region may have a wild-type sequence or a modified sequence. In some embodiments, the bispecific antibody comprises an engineered CH2 and / or engineered CH3 domain. In some cases, the modified heavy chain may comprise a modified Fc domain comprising: In this case, the modified Fc domain may use electrostatic steering effects, including For example, Gunasekeran et al, (2010) Journal of Bio Logical Chemistry 285, 19637-19646 (the disclosure of which is incorporated herein by reference) by use of the procedures described in (which is incorporated herein by reference in its entirety). In some cases, the bispecific antibody may comprise a CH3 domain. The heavy chains are mainly assembled by charge pair substitutions, e.g., K392D and K4 The other heavy chain contains E356K and D399K substitutions. The charge pair substituted chains may be modified to have the same or different structure as each other. The numbering of amino acid substitutions follows the EU numbering scheme of HC. Follow the stem.

[0122] In some cases, the antibodies of the present disclosure comprise charge pair substitutions. Thus, the antibodies of the present disclosure do not contain charge pair substitutions. Alternative means of promoting efficient heterodimer formation can be used.

[0123] In some cases, the modified heavy chain comprises a knob-into-hole "Knob-into-hole" amino acid modifications can be used in antibody engineering. Rational design strategies for the production of multispecific antibodies, including bispecific IgG antibodies For example, in the knob-into-hole battle, The antibody is incorporated into a bispecific antibody made from two monoclonal antibodies of different specificities. In this study, amino acid changes were engineered to create a “no” in the CH3 of the heavy chain of monoclonal antibody 1 (mAb1). A "hole" is created 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 can be represented by a small amino acid such as threonine (T). The into-hole modifications were a T22Y substitution in the first CH3 domain and a partner C A Y86T substitution can be made in the H3 domain. An example of a knob-into-hole modification is Carter er, J. Immunol. Methods, 248(1-2):7-15(2001) , Ridgway, JBet al.Protein Eng.9(7):617- 2 (1996), and Merchant, AM et al. Nat. Biotech Nol. 16(7):677-81 (1998), the disclosures of which are incorporated herein by reference. The present specification is incorporated in its entirety. Among antibodies generated from these, bispecific heterodimers generally represent the majority.

[0124] In certain embodiments, the bispecific antibodies provided herein bind to ABCC1 and TA A, while expressing ABCC1 and / or TAA. In other words, the dual antibodies provided herein exhibit reduced binding to non-cancer cells expressing the antibody. The specific antibody is (1) a cell that expresses a TAA and has low or no ABCC1 expression. and (2) cells expressing ABCC1 that have low or no TAA expression. ABCC1 and TAAs are absent, bind with low affinity to cells, and and one or both at relatively high levels, i.e., higher than normal cells. It binds to cancer cells with high affinity.

[0125] Compositions and Formulations The present disclosure provides compositions comprising a subject antibody. The subject antibody compositions comprise, in addition to the subject antibody: Salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers ng 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 Methylpropanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (M OPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (T APS); solubilizers; detergents, e.g., non-ionic detergents such as Tween-20; The composition may include one or more of: a soluble fiber; a soluble fiber; a soluble fiber inhibitor; glycerol; and the like.

[0126] 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" is an amount that is effective to achieve the desired result, e.g., to achieve a desired effect in a subject. to produce a reduction in cancer in subjects, a reduction in the rate of cancer growth in subjects, or an improvement in cancer symptoms. Generally, the desired result is at least a reduction in the incidence of cancer compared to a control. Reduction in symptoms, reduction in cancer growth, reduction in cancer size. The body may be delivered or formulated in such a way as to avoid the blood-brain barrier.

[0127] In some cases, the antibody may include a delivery enhancer, such as an enhancer that crosses the blood-brain barrier. For example, increased permeability may be achieved to allow efficient transdermal delivery. nothing.

[0128] In some cases, the antibodies of the present disclosure are administered in a formulation with a delivery-enhancing agent. In some cases, the antibodies of the present disclosure may not themselves be able to cross the blood-brain barrier. In some cases, the antibodies of the present disclosure may enhance the permeability of tumor cells to anti-tumor agents, e.g., As a delivery enhancer to facilitate crossing of the blood-brain barrier by immunotherapeutic or chemotherapeutic agents. In some cases, the antibodies of the present disclosure may be used in combination with other antibodies or chemotherapeutic agents, such as The active agent crosses the blood-brain barrier, the blood-cerebrospinal fluid (CSF) barrier, the blood-testis barrier, or the blood-placenta barrier. It can be used as a delivery enhancer to facilitate crossing of the barrier.

[0129] In the subject methods, the subject antibodies are any antibodies capable of producing a desired therapeutic or diagnostic effect. The agent can be administered to the host using any convenient means. More specifically, the subject antibodies can be incorporated into various formulations for administration. and formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent. It can be used in tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and air The preparations may be formulated in solid, semi-solid, liquid, or gaseous form, such as a sol.

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

[0131] The subject antibodies may be prepared from vegetable or other similar oils, synthetic fatty acid glycerides, higher fatty acid or is soluble in aqueous or non-aqueous solvents such as esters of propylene glycol, as required. together with conventional additives such as toners, isotonicity agents, suspending agents, emulsifying agents, stabilizers, and preservatives. They can be formulated into an injectable preparation by dissolving, suspending, or emulsifying them. .

[0132] Pharmaceutical compositions containing the subject antibodies may be prepared by purifying the antibodies with a desired purity, optionally in a physiologically acceptable form. Mix with acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or tonicity agents. Acceptable carriers, excipients, and / or stabilizers may be used in the formulation. Non-toxic to recipients at dosages and concentrations, such as phosphoric acid, citric acid, and other organic acids Buffering agents; contains ascorbic acid, glutathione, cysteine, methionine, and citric acid Antioxidants; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cre sol, p-chloro-m-cresol, methyl or propyl paraben, benzal chloride arginine, glycine, ornithine, lysine, hyaluronic acid, niacin ... Styrene, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenyl thiamin, tyrosine, tryptophan, methionine, serine, proline, and their Combinations of amino acids; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (approximately 10 residues) polypeptides such as gelatin or serum albumin; proteins such as EDTA chelating agents; trehalose, sucrose, lactose, glucose, mannose, maize Lutose, galactose, fructose, sorbose, raffinose, glucosamine, N -Sugars such as methylglucosamine, galactosamine, and neuraminic acid; and / or T Ween, Brij Pluronics, Triton-X, or polyethylene Non-ionic surfactants such as PEG (PEG-1).

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

[0134] Exemplary antibody concentrations in a subject pharmaceutical composition are from about 1 mg / mL to about 200 mg / mL, or About 50 mg / mL to about 200 mg / mL, or about 150 mg / mL to about 200 mg / mL The range may be:

[0135] The aqueous antibody formulation may be prepared in a pH buffer solution, for example, at a pH of about 4.0 to about 7.5, or about 5.0 to about 6.5. It may be prepared at a pH in the range of about 6.0, or alternatively about 5.5. Suitable pH values ​​within this range are Examples of suitable buffers include phosphate, histidine, citrate, succinate, and acetate buffers, and other organic acid buffers. The buffer concentration can be adjusted based on, for example, the buffer and the desired tonicity of the formulation. Depending on the concentration, the concentration may be about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0136] In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be used. The term "isotonic" refers to the amount of isotonicity to which it is compared, such as the physiological salt solution or serum. The isotonicity of the isotonic agent is about 5 mM to about 35 mM. It may be used in an amount of 0 mM, for example, in an amount of 100 mM to 350 nM.

[0137] Surfactants may also be added to antibody formulations to reduce aggregation of the formulated antibody and / or to facilitate production. These surfactants may minimize the formation of particulates in the formulation and / or reduce adsorption. Exemplary surfactants include: , polyoxyethylene sorbitan fatty acid ester (Tween), polyoxyethylene alkyl ether (Brij), alkylphenyl polyoxyethylene ether (Trit on-X, polyoxyethylene-polyoxypropylene copolymer (Poloxame r, Pluronic), and sodium dodecyl sulfate (SDS). Exemplary concentrations of the agent can range from about 0.001% to about 1% w / v.

[0138] Also, cryoprotectants may be added to prevent unstable active ingredients (e.g., proteins) from being lyophilized. These agents may protect against destabilizing conditions during processing. For example, known cryoprotectants include sugars (glucose, and sucrose), polyols (mannitol, sorbitol, and glycerol Contains niacin, niacin, niacin, niacin, niacin-containing fatty acids, and amino acids (including alanine, glycine, and glutamic acid) The cryoprotectant may be included in an amount of about 10 mM to 500 nM.

[0139] In some embodiments, the subject formulation comprises a subject antibody and a drug product as identified above. It contains one or more of the following (e.g., surfactants, buffers, stabilizers, isotonicity agents), ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or one or more of propylparaben, benzalkonium chloride, and combinations thereof In other embodiments, the preservative is essentially free of, for example, about 0.001 It is included in formulations at concentrations ranging from about 2% (w / v).

[0140] For example, the subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration, and can be about 1 mg / mL to about 200 mg / mL of the target antibody, about 0.001% to about 1% of at least a surfactant, about 1 mM to about 100 mM of a buffering agent, optionally about 10 mM to about 50 mM of a The solution may contain from about 4.0 to about 50 mM of a stabilizer, and from about 5 mM to about 305 mM of an isotonicity agent. It has a pH of 7.0.

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

[0142] As used herein, the term "unit dosage form" refers to a dosage form for human and animal subjects. refers to physically discrete units suitable as unit dosages, each unit being a pharmaceutically acceptable diluent. A predetermined amount calculated in association with an agent, carrier, or vehicle in an amount sufficient to produce a desired effect. The specifications for the subject antibody depend on the particular antibody used and the results achieved. The efficacy of each antibody may depend on the intended effect, as well as the pharmacodynamics associated with each antibody in the host.

[0143] The subject antibodies can be administered as an injectable formulation. Typically, the injectable formulation The compositions may be prepared as liquid solutions or suspensions, and may be dissolved or suspended in a liquid vehicle prior to injection. Solid forms suitable for administration may also be prepared. The preparation may also be emulsified, or the antibody may be placed in a liposome. The compound may be encapsulated in a vehicle.

[0144] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Additionally, optionally, the vehicle may be They may also contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, or pH buffering agents. Actual methods of preparation will be known or apparent to those skilled in the art.

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

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

[0147] Dosage The appropriate dosage will be determined by your physician or other qualified medical professional based on a variety of clinical factors. As is well known in the medical field, the amount of time that can be spent for any one patient can be determined by the practitioner. The dosage of the drug depends on the patient's size, body surface area, age, the specific compound being administered, the patient's sex, and the duration of the Many factors, including the duration and route of administration, general health, and other medications being administered at the same time. The antibody of interest may be administered at a dose of 1 ng / kg body weight to 20 mg / kg body weight, e.g., 0 .1mg / kg body weight to 10mg / kg body weight, e.g. 0.5mg / kg body weight to 5mg / kg The amount of the active ingredient may be administered in amounts of body weight, but may be below or below this exemplary range, particularly taking into consideration the factors mentioned above. If the regimen is a continuous infusion, doses of 1 kg of body weight per minute are envisaged. It can also be in the range of 1 μg to 10 mg per unit.

[0148] Those skilled in the art will appreciate that dosage levels will vary depending on the particular antibody, the severity of the symptoms, and the subject's susceptibility to side effects. It will be readily understood that the preferred Suitable dosages are readily determinable by those of skill in the art by a variety of means.

[0149] Route of administration The subject antibodies can be administered by a variety of pharmaceutical, therapeutic, and therapeutic methods, including in vivo and ex vivo methods, and systemic and local routes of administration. The compound may be administered to an individual using any available method and route suitable for drug delivery.

[0150] Conventional pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, and topical. Applications include intravenous, intra-arterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. The routes of administration may be combined or combined as needed depending on the antibody and / or the desired effect. The subject antibody compositions can 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. The compositions are administered locally. In some embodiments, a subject antibody composition is administered intracranially. In some embodiments, a subject antibody composition is administered intravenously.

[0151] The agent may be administered by any available route suitable for conventional drug delivery, including systemic or local routes. Conventional methods and routes of administration can be used to administer to the host. Contemplated routes of administration include, but are not necessarily, enteral, parenteral, or inhalation routes. Not limited to.

[0152] Parenteral routes of administration other than inhalation include topical, transdermal, subcutaneous, intramuscular, intraorbital, intracisternal, and spinal. This includes intravenous, intrasternal, and intravenous routes, i.e., any route of administration other than through the gastrointestinal tract. Parenteral administration can result in systemic or local delivery of the subject antibody. When systemic delivery is desired, administration is typically performed in a pharmaceutical preparation. It involves topical or mucosal administration of the product, which is then absorbed invasively or systemically.

[0153] The subject antibodies can also be delivered to a subject by enteral administration. Enteral routes of administration include: including, but not necessarily limited to, oral and rectal (e.g., using a suppository) delivery do not have.

[0154] Treatment means at least amelioration of symptoms associated with a pathological condition afflicting the host; Improvement is used in a broad sense to refer to the reduction of cancer and / or cancer growth and associated pain. and reducing the magnitude of a parameter associated with the pathological condition being treated, e.g., symptoms. Therefore, treatment also refers to the reduction of a pathological condition, or at least the reduction of the symptoms associated therewith. The symptoms are completely inhibited, e.g., prevented from occurring, or stopped, e.g., terminated. whereby the host is no longer in a pathological state, or at least not characterized by a pathological state. This includes situations where the patient does not suffer from symptoms associated with the condition.

[0155] Various subjects (the term "subject" is used herein in conjunction with "individual" and "patient"). (used interchangeably with the term 'anti-inflammatory drug') are treatable according to the methods of the present disclosure. Elephants are "mammals" or "mammals," and these terms refer to animals in the order Carnivora (e.g., dogs). and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., broadly used to describe organisms within the class Mammalia, including, for example, humans, chimpanzees, and monkeys In some embodiments, the host is a human.

[0156] Kits with unit doses of the subject antibodies are provided, e.g., oral or injectable doses. In some embodiments, in addition to the container containing the unit dose, a target pathological There is an informational package insert that explains the use of the antibody in treating the condition and the associated benefits.

[0157] nucleic acid The present disclosure provides nucleic acids comprising nucleotide sequences encoding the subject antibodies. The encoding nucleotide sequence is then delivered to the intended target cell (e.g., the cell that synthesizes the encoded antibody). and / or secretion of the nucleotide sequence in cells can be manipulated to one or more regulatory elements, such as promoters and enhancers, that allow can be linked to

[0158] Suitable promoter and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include lacI, lacZ, T3, These include, but are not limited to, T7, gpt, lambda P, and trc. For expression in biological cells, suitable promoters include promoters for light and / or heavy chain immunoglobulins. phosphogene promoter and enhancer elements, cytomegalovirus immediate early promoter promoter, herpes simplex virus thymidine kinase promoter, early and late SV4 0 promoter, promoters present in long terminal repeats from retroviruses, mouse The tarothionine-I promoter, as well as various art-known tissue-specific promoters, These include, but are not limited to:

[0159] Nucleotide sequences encoding the subject antibodies are contained in expression vectors and / or cloning vectors. If the subject antibody comprises two or more distinct polypeptides, The nucleotide sequences encoding the two polypeptides may be cloned into the same or separate vectors. The separate polypeptides can be generated by separate promoters, one or more internal ribosome entry site (IRES), one or more self-cleaving sequences (e.g., P2A, T2A, various strategies, such as E2A and F2A cleavage sequences, and combinations thereof. Thus, the expression vector can be expressed from a single nucleic acid or a single vector. The vector may include a vector sequence, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Cut.

[0160] Large numbers of suitable vectors and promoters are known to those of skill in the art, and many are suitable for use in recombinant constructs of interest. The following vectors are provided as examples: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagen e, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala) a,Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1 , pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (P harmacia).

[0161] Expression vectors generally have convenient restriction sites located near the promoter sequence. The insertion of a nucleic acid sequence encoding a heterologous protein is provided. Suitable expression vectors include viral vectors (e.g., viral vectors), Viral vectors based on poliovirus, adenovirus, adenovirus Related viruses: SV40, herpes simplex virus, human immunodeficiency virus, retrovirus vectors (e.g., murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus) virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative vectors derived from retroviruses such as human sarcoma virus and mammary tumor virus) These include, but are not limited to:

[0162] Nucleic acids, e.g., as described herein, can be used in some cases, e.g., to transfect cells with nucleic acids. The nucleic acid can be introduced into cells by contacting the cells with the nucleic acid. Generally, these are referred to herein as genetically modified cells. A variety of nucleic acid delivery methods can be used, including: For example, naked nucleic acid delivery, viral delivery, chemical transfection, gene guns, etc. This includes, but is not limited to:

[0163] cell The present disclosure provides isolated genetically modified cells (e.g., in vitro) that are genetically modified with a nucleic acid of interest. In some embodiments, the present invention provides a method for producing a cell line comprising administering to a subject ... an in vitro cell, an ex vivo cell, a cultured cell, or the like. In some cases, the isolated genetically modified cells of interest are capable of producing the antibody of interest. In the method, the genetically modified cells can deliver the antibody, for example, to a subject in need thereof. can.

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

[0165] Suitable mammalian cells include primary cells and immortalized cell lines. include human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include 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 These include, but are not limited to, cells.

[0166] In some cases, useful mammalian cells include cells derived from mammalian tissues or organs. In some cases, the cells used are kidney cells, e.g., HEK These include kidney cells from established kidney cell lines such as 293T cells.

[0167] In some cases, the cells of the present disclosure may be immune cells. When used in this context, the term "immune cells" generally refers to 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, and dendritic cells). "T cells" include T helper cells (C D4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and cancer This includes all types of immune cells that express CD3, including MA-delta T cells. The "toxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils. These cells are capable of mediating a cytotoxic response.

[0168] In some cases, useful cells expressing antibodies, such as the multispecific antibodies of the present disclosure, These may include producer T cells. In some cases, engineered producer T cells can deliver antibodies to the target cells, which then need them. It can be used to deliver to a subject.

[0169] In some cases, the immune cells of the present disclosure comprise an ABCC1 binding domain, a transmembrane domain, and an immune enzyme comprising a chimeric antigen receptor (CAR) containing an intracellular signaling domain. The vector cells contain the ABCC1 binding domain of the variable heavy chain ( The heavy chain complementarity determining region (HCDR) and light chain C of the pair of variable (VH) and variable light (VL) chains In one embodiment, the intracellular signaling domain comprises at least and / or one or more costimulatory molecules, such as 4-1BB (i.e., CD137), CD27, and / or Alternatively, it may contain one or more functional signaling domains derived from CD28. The null transduction domain is a functional signaling domain derived from a costimulatory molecule and a stimulatory molecule. It may comprise a functional signaling domain derived from

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

[0171] method As summarized above, the methods of the present disclosure include contacting a cell with an antibody of the present disclosure, and a method of treating a subject according to the methods described in this application, which method involves administering an antibody of the present disclosure to the subject. and methods for producing components, such as antibodies, compositions and formulations, nucleic acids, expression vectors, cells, etc. Including the law.

[0172] As summarized above, the methods of the present disclosure involve contacting cancer cells with an antibody of the present disclosure to, e.g., For example, the presence of ABCC1 expression on cancer cells can be detected, and the level of ABCC1 expression on cancer cells can be analyzed. These include measuring the level of cancer cells, or promoting and / or enhancing the killing of cancer cells. In this case, the killing of cancer cells is due to immune responses acting on cancer cells that are bound by antibodies. In some cases, cancer cell killing is mediated by immune cells, e.g. For example, antibody-mediated ABCC1 inhibition results in inhibition of tumor cell efflux. In some cases, cancer cell killing is achieved by inhibiting the cellular elimination of cancer cells and by inhibiting immune responses. The immune response is mediated by a combination of immune-mediated responses (e.g., via the Fc region of an antibody). Methods involving contacting cancer cells with an antibody of the present disclosure can include treating the cancer cells with, for example, chemotherapy, may include contacting with additional therapies or active agents, including immunotherapy, radiation therapy, etc. Or it cannot be included.

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

[0174] Thus, administration can include, for example, delivery of the antibody by injection, delivery of the antibody by infusion, administration of the antibody delivery of a nucleic acid or expression vector encoding the antibody to a subject; administration of cells that express and secrete the antibody to a subject. delivery of antibodies by immobilizing the ABCC1-binding domain, transmembrane domain, and Immune effectors expressing chimeric antigen receptors (CARs) containing intracellular signaling domains These include, but are not limited to, delivery of tumor cells (e.g., CAR-T cells), ABC The C1 binding domain comprises the HCDRs and VL domains of pairs of antibodies listed in Table 2. The administration of a drug, a nucleic acid encoding a drug, or a cell expressing a drug may include: This may include contacting with an agent, contacting with a nucleic acid, contacting with a cell, and the like.

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

[0176] In some embodiments, an effective amount of a subject antibody is administered alone (e.g., in monotherapy). in combination with one or more additional therapeutic agents (e.g., in combination therapy) which, when administered in one or more doses, ameliorates cancer in the individual being treated. (i.e., slowing cancer growth, stopping cancer growth, reversing cancer growth, For example, an effective amount of the target The antibody may be at least about 5%, at least about 10%, or , 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, to reduce the rate of cancer growth in the individual. It may reduce or decrease the size of the cancer in an individual.

[0177] In some cases, the subject may be treated systemically, administering the subject antibody in combination with one or more additional "Systemic treatment" as used herein includes the use of In some cases, the tumor may be a specific tumor (e.g., a primary tumor or a defined secondary tumor) or a specific cancer. targeting the tissue involved (e.g., liver in cases of liver cancer, blood in cases of blood cancer, etc.) Systemic treatment generally refers to treatment that is directed at the subject's body as a whole, e.g. For example, this may include systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof, etc. However, it is not limited to these.

[0178] In some cases, the subject may be treated locally, administering the subject antibody in combination with one or more additional "Topical treatment" as used herein includes the use of In this case, it is specifically directed to the location of the tumor (e.g., the primary tumor or a defined secondary tumor). or tissue containing cancer (e.g., liver in cases of liver cancer, In some cases, localized treatment is used. Therapy also involves the environment surrounding the tumor, such as the tissue immediately adjacent to the tumor, surrounding the tumor, and Localized treatments generally involve administering a therapeutic agent to the site of a tumor, such as a primary tumor. The antibody of interest does not affect or target tissues distant from the cancer site, including the tumor site. Useful local treatments that can be administered in addition to or in combination with the subject antibodies include, for example, hand Surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, or a combination thereof This includes, but is not limited to:

[0179] In some embodiments, a subject treatment method comprises administering a subject antibody and one or more additional therapeutic agents Suitable additional therapeutic agents include chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, and the like. These include, but are not limited to, therapeutic reagents, other antibody agents, etc. Additional therapies that may be administered to a subject before, during, or after treatment may depend, for example, on the type of cancer, the subject's condition, Varies depending on many factors, including history, general health, and / or any co-morbidities Useful cancer therapies include, for example, radiation therapy, chemotherapy, and immunotherapy. These include, but are not limited to:

[0180] Radiation therapy can involve the delivery of radiation from an externally applied source such as a beam or small source of radiation. These include, but are not limited to, x-rays or gamma rays delivered by an implant.

[0181] Antibodies suitable for use in cancer therapy include naked antibodies, such as trastuzumab ( Herceptin), bevacizumab (Avastin™), cetuximab (Er bitux™), panitumumab (Vectibix™), ipilimumab (Y ervoy™), rituximab (Rituxan), alemtuzumab (Lemtr ada™), ofatumumab (Arzerra™), oregovomab (Ova Rex™), lambrolizumab (MK-3475), pertuzumab (Perjet a™), ranibizumab (Lucentis™), etc., and conjugated Antibodies, such as gemtuzumab ozogamicin (Mylortarg™), Brent Ximab vedotin (Adcetris™), 90Y-labeled ibritumomab tiuximab Tan (Zevalin™), 131I-labeled Tositumoma (Bexxar™) These include, but are not limited to:

[0182] Antibodies suitable for use in cancer therapy also include antibodies raised against tumor-associated antigens. Such antigens include, but are not limited to, CD20, CD30, CD33, CD5 2, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, leaf Acid-binding proteins, 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, EPHA 3, 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, E These include, but are not limited to, SR1, CTLA-4, LAG-3, and TIM-3. These antibodies may be used in combination therapy with the anti-ABCC1 antibodies provided herein. It can be administered as follows.

[0183] Conventional cancer therapies also include targeted therapies for cancer, such as HER2 (ER Ado-trastuzumab emtansine (Kadcyla) targets BB2 / neu (approved for use in breast cancer), EGFR (HER1 / ERBB1), HER2 ( Afatinib (Gilotrif), which targets ERBB2 / neu (non-small cell lung cancer) approved for use in the kidney), targeted aldesleukin (Proleukin) approved for use in cancers such as melanoma, and alectinib, which targets ALK (Al ecensa (approved for use in non-small cell lung cancer), an antibody targeting CD52 Lemtuzumab (Campath) (approved for use in B-cell chronic lymphocytic leukemia) ), and atezolizumab (Tecentriq), which targets PD-L1 (urothelial cancer, non- approved for use in small cell lung cancer), and avelumab (Bav encio) (approved for use in Merkel cell carcinoma), KIT, PDGFRβ, Axitinib (Inlyta), which targets VEGFR1 / 2 / 3 (used in renal cell carcinoma) and belimumab (Benlysta) (approved for rheumatoid arthritis), which targets BAFF. approved for use in rheumatoid arthritis, and belinostat, which targets HDACs odaq) (approved for use in peripheral T-cell lymphoma), which targets VEGF ligands Bevacizumab (Avastin) (cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma) , approved for use in non-small cell lung cancer, ovarian cancer, peritoneal cancer, and renal cell cancer), CD Blincyto (Blincyto), which targets 19 / CD3 (acute lymphoblastic leukemia) approved for use in precursor B cell disease, and bortezomib, which targets the proteasome Velcade (approved for use in multiple myeloma and mantle cell lymphoma) and bosutinib (Bosulif), which targets ABL (for use in chronic myeloid leukemia). approved), brentuximab vedotin (Adcetris), which targets CD30 ) (approved for use in Hodgkin's lymphoma and anaplastic large cell lymphoma), targeting ALK Brigatinib (Alunbrig), approved for use in non-small cell lung cancer (ALK+), (approved), Caboza, which targets FLT3, KIT, MET, RET, and VEGFR2 entinib (Cabometyx, Cometriq) (in medullary thyroid cancer, renal cell carcinoma) approved for use), and the proteasome-targeting carfilzomib (Kyprol is) (approved for use in multiple myeloma), ceritinib (Z ykadia) (approved for use in non-small cell lung cancer), EGFR (HER1 / E Cetuximab (Erbitux) targeting RBB1 (colorectal cancer, squamous cell carcinoma of the head and neck) approved for use in epithelial cancers), and cobimetinib (Cotell ic) (approved for use in melanoma), a clone targeting ALK, MET, and ROS1 Rizotinib (Xalkori) (approved for use in non-small cell lung cancer), BRAF Dabrafenib (Tafinlar) targets the α-amyloid receptor β (α-amyloid) and is used in melanoma and non-small cell lung cancer. approved), daratumumab (Darzalex), which targets CD38 (multiple bone marrow transplants) approved for use in myeloma), dasatinib (Sprycel), which targets ABL ( approved for use in chronic myeloid leukemia, acute lymphoblastic leukemia, and Targeted denosumab (Xgeva) (approved for use in giant cell tumor of bone), B 4Dinutuximab (Unituxin) targeting GALNT1 (GD2) (pediatric neurology) approved for use in blastoma), and durvalumab (Imf inzi) (approved for use in urothelial carcinoma), SLAMF7 (CS1 / CD3 elotuzumab (Empliciti) (multiple myeloma) targeting 19 / CRACC approved for use in acute myeloid leukemia (ARDS), and enasidenib (Idhifa), which targets IDH2 ( approved for use in myeloid leukemia), targeting EGFR (HER1 / ERBB1) Erlotinib (Tarceva), approved for use in non-small cell lung cancer and pancreatic cancer everolimus (Afinitor), which targets mTOR (pancreatic, gastrointestinal, or pulmonary Neuroendocrine tumors of origin, renal cell carcinoma, unresectable subependymal giant cell astrocytoma, breast cancer approved for use), gefitinib, which targets EGFR (HER1 / ERBB1) (Iressa) (approved for use in non-small cell lung cancer), which targets CD20 Ibritumomab tiuxetan (Zevalin) (approved for use in non-Hodgkin's lymphoma) ibrutinib (Imbruvica), which targets BTK (mantle cell lymphoma) Approved for use in lymphoma, chronic lymphocytic leukemia, and Waldenström's macroglobulinemia idelalisib (Zydelig), which targets PI3Kδ (chronic lymphocytic leukemia) Approved for use in leukemia, follicular B-cell non-Hodgkin's lymphoma, and small lymphocytic lymphoma imatinib (Gleevec) (GI Approved for use in stromal tumors (KIT+), dermatofibrosarcoma protuberans, and multiple hematological malignancies ipilimumab (Yervoy), which targets CTLA-4 (approved for use in melanoma), approved), and proteasome-targeting ixazomib (Ninlaro) (multiple approved for use in myeloma), HER2 (ERBB2 / neu), EGFR (HE Lapatinib (Tykerb) targets R1 / ERBB1 (breast cancer (HER2+)) approved for use in pediatric patients), and lenvatinib (Lenvima), which targets VEGFR2. ) (approved for use in renal cell carcinoma and thyroid cancer), and Midos, which targets FLT3 Taurine (Rydapt) (approved for use in acute myeloid leukemia (FLT3+) ), and necitumumab (Portrazza), which targets EGFR (HER1 / ERBB1). ) (approved for use in squamous non-small cell lung cancer), HER2 (ERBB2 / ne u)-targeting neratinib (Nerlynx) (approved for use in breast cancer); Nilotinib (Tasigna), which targets ABL (approved for use in chronic myeloid leukemia), and niraparib (Zejula), which targets PARP (ovarian cancer, fallopian tube cancer, approved for use in peritoneal cancer), and nivolumab (Opdivo), which targets PD-1. ) (colorectal cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, melanoma, non-small cell lung cancer, (approved for use in renal cell carcinoma and urothelial carcinoma), and Obinutr, which targets CD20 Izabu (Gazyva) (approved for use in chronic lymphocytic leukemia and follicular lymphoma) ofatumumab (Arzerra, HuMax-CD20), which targets CD20 (approved for use in chronic lymphocytic leukemia), and olaparib, which targets PARP ( Lynparza (approved for use in ovarian cancer), which targets PDGFRα Lartruvo (approved for use in soft tissue sarcomas), EGF Osimertinib (Tagrisso), which targets R (approved for use in non-small cell lung cancer), palbociclib (Ibrance), which targets CDK4 and CDK6 (breast cancer) approved for use in cancer), and pancreatic cancer targeting EGFR (HER1 / ERBB1). tumab (Vectibix) (approved for use in colorectal cancer), which targets HDAC Panobinostat (Farydak), approved for use in multiple myeloma, is a ), pazopanib (Votrient), which targets VEGFR, PDGFR, and KIT (renal approved for use in cell carcinomas), and pembrolizumab (Key Truda) (classical Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+), head and neck cancer Approved for use in cervical squamous cell carcinoma, solid tumors (MSI-H), HER2 (E Pertuzumab (Perjeta) targets RBB2 / neu (breast cancer (HER2+) ), targeting ABL, FGFR1-3, FLT3, and VEGFR2 Ponatinib (Iclusig) (chronic myeloid leukemia, acute lymphoblastic leukemia) approved for use in patients with glaucoma, and ramucirumab (Cyramza), which targets VEGFR2 (For use in colorectal cancer, gastric cancer or esophagogastric junction (GEJ) adenocarcinoma, and non-small cell lung cancer) approved), targeting KIT, PDGFRβ, RAF, RET, and VEGFR1 / 2 / 3 Regorafenib (Stivarga) targets colorectal cancer, gastrointestinal stromal tumors, and hepatocellular carcinoma. approved for use in rheumatoid arthritis, and ribociclib (Kis qali) (approved for use in breast cancer (HR+, HER2-)), targeting CD20 Rituximab (Rituxan, Mabthera) (non-Hodgkin's lymphoma, chronic (approved for use in chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis) , CD20-targeted rituximab / hyaluronidase human (Rituxan Hyc ela) (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma) approved for use in the skin), romidepsin (Istodax), which targets HDACs approved for use in T-cell lymphoma, peripheral T-cell lymphoma, and PARP-targeting rucaparib (Rubraca) (approved for use in ovarian cancer), JAK1 / 2 Ruxolitinib (Jakafi), which targets β-glucan (approved for use in myelofibrosis) , IL-6-targeting siltuximab (Sylvant) (multicentric Castleman disease) approved for use in the US), and the targeted sipuleucel-T (Provenge) approved for use in prostate cancer), and sonidegib (Odomzo), which targets smooth muscle. (approved for use in basal cell carcinoma), VEGFR, PDGFR, KIT, RAF sorafenib (Nexavar) (hepatocellular carcinoma, renal cell carcinoma, thyroid cancer) approved for use in the treatment of mTOR-targeted rheumatoid arthritis (rheumatoid arthritis), and temsirolimus (Torisel), which targets mTOR ( approved for use in renal cell carcinoma), and tositumomab (Bexx), which targets CD20 ar) (approved for use in non-Hodgkin's lymphoma), tramethicone, which targets MEK Nib (Mekinist) (approved for use in melanoma and non-small cell lung cancer), HE Trastuzumab (Herceptin) targets R2 (ERBB2 / neu) approved for use in gastric cancer (HER2+), EGFR ( Vandetanib (Caprels) targets R1 / ERBB1, RET, and VEGFR2 a) Vemurafenib, which targets BRAF (approved for use in medullary thyroid cancer) Zelboraf (approved for use in melanoma), a vector that targets BCL2 Netoclax (Venclexta) (approved for use in chronic lymphocytic leukemia) , PTCH, and vismodegib (Erivedge) targeting smooth muscle (in basal cell carcinoma) approved for use in the treatment of HDAC-related cancers, and vorinostat (Zolinza) ( Approved for use in cutaneous T-cell lymphoma), PIGF, and VEGFA / B Ziv-aflibercept (Zaltrap) (approved for use in colorectal cancer) These antibodies include, but are not limited to, the antibodies provided herein. It may be administered as a combination therapy with an ABCC1 antibody.

[0184] Suitable biological response modifiers for use in connection with the methods of the present disclosure include: (1) tyrosine kinases (2) inhibitors of serine / threonine kinase activity; (3) inhibitors of tumor (4) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; 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- Chemotherapeutic agents are compounds of the cytotoxic and cytostatic nature. Non-limiting examples of include alkylating agents (e.g., nitrosoureas), antimetabolites (e.g., methotrexate), antitumor antibiotics (e.g., anthracyclines), plant alkaloids amides (e.g., vinca alkaloids, taxanes, etc.), toposiomerase inhibitors, and steroids Contains steroid hormones.

[0186] Agents that act to reduce cell proliferation are known and widely used in the art. Such agents include nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkylating agents such as alkyl sulfonates and triazenes; mechlorethamine; cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), Carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) , streptozocin, chlorozotocin, uracil mustard, chlormethine, ifospha Mido, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoric acid These include, but are not limited to, ramimin, busulfan, dacarbazine, and temozolomide. I can't.

[0187] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine dehydrogenases. These include enzyme inhibitors, such as cytarabine (CYTOSAR-U), cytosine arabinoside, and 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, fludanab phosphate These include, but are not limited to, rabin, 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®), dextromethorphan (DDT), Dixicoformycin, mitomycin-C, L-asparaginase, azathioprine; Alkaloids, such as vincristine, vinblastine, vinorelbine, Vindesine, etc.; Podophyllotoxins, e.g., etoposide, teniposide, etc.; Antibiotics , e.g., anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomicin cerubicin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives Phenoxyzomides, cyclopeptides, for example, dactinomycin; basic glycopeptides anthraquinone glycosides, such as plicamycin ( Mithramycin); anthracenediones, e.g., mitoxantrone; azirinopyrroloi andregiones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, These include FK-506 (tacrolimus, Prograf), rapamycin, etc. Not limited.

[0189] Other antiproliferative cytotoxic agents include navelbene, CPT-11, anastrazole, letrazole, zole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and dolomide It is Xafin.

[0190] Microtubule-affecting agents with antiproliferative activity are also suitable for use, including but not limited to, allococcal Luchtin (NSC 406042), Halichondrin B (NSC 609395), Col Colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dorsal Tatin 10 (NSC 376128), Maytansine (NSC 153858), Rhizox Shin (NSC 332598), paclitaxel (Taxol®), Taxo 1 (registered trademark) derivatives, docetaxel (Taxotere (registered trademark)), thiocolchic (NSC 361792), Tritylcysterol, Vinblastine Sulfate, Vincris thiamin sulfate, natural and synthetic epothilones (etopirone A, epothilone B, discodermo including but not limited to rifampin, estramustine, and nocodazole.

[0191] Suitable hormone regulators and steroids (including synthetic analogs) for use include adrenocortical hormones, steroids, e.g., prednisone, dexamethasone, and the like, estrogen and prednisone; Progesterones, e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate adrenal glands, such as steroids, megestrol acetate, estradiol, clomiphene, and tamoxifen Cortical depressants, such as aminoglutethimide, 17α-ethinylestradiol, diethyl Rustilbestrol, testosterone, fluoxymesterone, dromos propionate Tanolone, testolactone, methylprednisolone, methyl-testolone, prednisolone Lon, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglucan Tethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamivir These include fluticasone (Drogenil), toremifene (Fareston), and Zoladex. Estrogen stimulates proliferation and differentiation, and therefore, Compounds that bind to the receptor are used to block this activity. can inhibit T cell proliferation.

[0192] Other chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazines. Drazine; Epidophyllotoxin; Topoisomerase inhibitor; Procarbazine; Mitoxa Other antiproliferative agents of interest include immunosuppressants such as leucovorin; tegafur; and the like. immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxyspergualin, azas Porin, leflunomide, mizoribine, azaspirane (SKF105685); Ires sa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)- 7-Methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline, etc. .

[0193] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "paclitaxel" (e.g., docetaxel, TAXOL™, TA XOTERE™ (docetaxel formulation), a 10-desacetyl derivative of paclitaxel analogues, and 3'N-desbenzoyl-3'Nt-butoxycarbonyl of paclitaxel (The term "antibody" should be understood herein to include analogs, preparations, and derivatives such as analogs.) are known to those skilled in the art (WO94 / 07882, WO94 / 07881, WO94 / 0 7880, WO94 / 07876, WO93 / 23555, WO93 / 10076, U.S. Patent Nos. 5,294,637, 5,283,253, and 5,279,949, Same No. 5,274,137, Same No. 5,202,448, Same No. 5,200,534, Same No. See also EP 590,267 and EP 5,229,529. or commercially available from, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia, or Taxus yan These compounds can be obtained from a variety of commercial sources, including T-1912 from T. nanensis.

[0194] Paclitaxel is available in the common chemically available form as well as in its analogues. analogs and derivatives (e.g., Taxotere™ docetaxel, as described above), and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel- Dextran, paclitaxel-xylose, or Abraxane® It should be understood to refer to protein-bound paclitaxel.

[0195] The term "taxane" refers to a variety of known compounds, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include those described in International Patent Application No. 99 / 18113. galactose and mannose derivatives, piperazines as described in WO99 / 14209 and other derivatives, WO99 / 09021, WO98 / 22451, and U.S. Pat. taxane derivatives described in WO98 / 28288; Thio derivatives, sulfenamide derivatives as described in U.S. Pat. No. 5,821,263, and and taxol derivatives described in U.S. Pat. No. 5,415,869, which Further included are prodrugs of paclitaxel, and are not limited to those disclosed in WO98 / 58927 , WO98 / 13059, and U.S. Pat. No. 5,824,701. But not limited to these.

[0196] Useful immunotherapies include anti-PD-1 / PD-L1 immunotherapy, and / or Immune checkpoint receptors such as CTLA-4, LAG-3, and TIM-3 can be targeted. Other immunotherapies include those targeting tumor-associated markers such as anti-PD-1 / PD-L1. Immunotherapy includes, for example, administering to a subject an effective amount of one or more anti-PD-1 / PD-L1 therapeutic agents. These include, but are not limited to, those therapies that involve administering a steroid or other agonist to Examples of antagonists include OPDIVO® (nivolumab), KEYTR UDA® (pembrolizumab), Tecentriq™ (atezolizumab) b), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559(MDX-1105), CA-170, BMS-202, BMS- These include, but are not limited to, BMS-8, BMS-37, BMS-242, etc. The antibodies can be administered as a combination therapy with the anti-ABCC1 antibodies provided herein. .

[0197] CTLA-4, also known as CD152, binds to CD80 and CD86. Antibodies against LA-4 have been approved to treat several cancer types. The co-inhibitory effect of CTLA-4 with other immunotherapies may allow CTLA-4 to be used in combination with other immunotherapies to treat certain cancers. TIM-3 also inhibits some can be targeted for immunotherapy in various cancer types.

[0198] LAG-3 is in clinical trials to treat cancer. Anti-LAG-3 immunotherapy includes: Activates T effector cells (transmits LAG-3 inhibitory signals to pre-activated LAG-3+ cells) down-regulating induced (i.e., antigen-specific) Treg suppressive activity Useful methods include those using antagonistic LAG-3 antibodies that can inhibit both LAG-3 antagonist antibodies include leratolimab (BMS-986016, Bristol-Myers Squibb). Developed by Immutep TSR-033 (anti-LAG-3 mAb, developed by TESARO, Inc.) (developed by

[0199] Immunotherapies also include, for example, adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) For example, the subject may be treated with a T cell-based immunotherapy, such as a T cell therapy, for example, a cancer-associated A population of CAR T cells engineered to target antigens expressed by the CAR can be administered. T cell-based therapies may, in some cases, be performed using a blood sample or tumor biopsy from a subject. and obtaining a cell sample of the immune cells from the sample, culturing the immune cells ex vivo, and For example, immune cells may be cultured with or without genetic modification. These cells were obtained from the tumor, cultured ex vivo, and modified with a CAR specific for an antigen expressed by the tumor. The CAR T cells can then be reintroduced into the subject. T cell-based immunotherapy can be used to target cancer, depending on the specific cancer being treated. to harvest / cultivate different cell types, for example, by targeting different antigens. In addition, T cell-based immunotherapy can be configured in various ways, e.g., by For example, by intravenous injection or locally, for example, by injection (e.g., intraperitoneal injection, pleural catheter). It can be administered systemically by catheter injection, direct injection, or the like.

[0200] In some cases, the treatment methods described herein include administering to a subject a multidrug-resistant transformant. transporters, including, but not limited to, multidrug resistance transporters other than ABCC1 The method may involve administering one or more inhibitors of multidrug resistance transporters, including but not limited to: Useful inhibitors include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and Inhibitors of multidrug resistance transporters include ABC transporters and small molecule inhibitors. These include acetylcholinesterase inhibitors.

[0201] Suitable individuals for treatment using the methods of the present disclosure include individuals with cancer, individuals diagnosed with cancer, Individuals who have been diagnosed with cancer or are being treated for cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc. , to treat the cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) Individuals who have been treated but are not responding to treatment, those who have had cancer treated with chemotherapy, radiation therapy, have been treated with one or more of the following: antibody therapy, surgery, etc., but have initially responded to treatment but This includes individuals who subsequently relapse, i.e., whose cancer has returned.

[0202] The disclosed methods can be used to treat, for example, primary cancer, secondary cancer, regrowing cancer, recurrent cancer, refractory cancer, These drugs can be used to target and treat a variety of cancers, including cancers of the genitals, liver, and kidney. In some cases, the disclosed methods may 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 in other than primary (e.g., secondary or or later), for example, in subjects with cancer that is refractory to previous treatments, In subjects with cancer that has regrown after prior treatment, a mixed response to prior treatment (e.g., a positive response to at least one tumor in the subject and at least one tumor in the subject and in subjects with a negative or neutral response to a second tumor.

[0203] In some cases, the methods of the present disclosure are directed to patients with drug-resistant cancer, such as multidrug-resistant cancer. Multidrug resistance (MDR) is a condition in which many cancers are resistant to chemotherapy. This is a mechanism by which resistance develops to drugs, resulting in minimal cell death and the expansion of drug-resistant tumors. MDR cancers may involve one or more resistance mechanisms, e.g., increased efflux pump activity. increased expression, decreased absorption of drugs, inhibition of cell death or apoptosis, modulation of drug metabolism, etc. In some cases, the methods of the present disclosure include, but are not limited to, preventing MDR. It can be stopped, reversed, or avoided.

[0204] In some cases, the methods of the disclosure include administering to a patient having a cancer that is resistant to the first agent. A subject receiving an effective amount of the compounds described herein in combination with a second agent that is different from the first agent. For example, in some cases, Thus, the subject's cancer may be resistant to a first chemotherapy, and the subject may be receiving a second chemotherapy that is different from the first. administering an effective amount of a subject antibody as described herein in combination with chemotherapy. Various combinations of first and second chemotherapeutic agents can be used, e.g. For example, 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, In particular, the methods of the present disclosure may find use in the treatment of various cancers, for example, acute lymphoblastic leukemia. Myeloid leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, and AIDS-related Cancer (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendix cancer, astrocytoma, atypical leukemia Teratoma / rhabdoid tumor, basal cell carcinoma, bile duct cancer (extrahepatic), bladder cancer, bone cancer (e.g. Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brainstem gliomas, brain tumors (e.g. For example, astrocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, epithelioma, etc. Breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumors, Kitt lymphoma, carcinoid tumors (e.g., pediatric, gastrointestinal), carcinoma of unknown primary, Cardiac (heart) tumors, central nervous system (e.g., atypical teratogenic / rhabdoid tumors, embryonic tumors, germ cell tumors) tumors, lymphomas, etc.), cervical cancer, childhood cancer, myeloma, chronic lymphocytic leukemia (CLL) ), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, cranial Pharyngoma, cutaneous T-cell lymphoma, ductal (e.g., bile duct, extrahepatic, etc.), ductal carcinoma in situ (D CIS), embryonal tumors, endometrial cancer, epithelioma, esophageal cancer, neuroepithelioma, Ewing's sarcoma, Extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), fibrous histiocytoma of bone (e.g., malignant, osteosarcoma, etc.), gallbladder cancer, stomach (Gastric) (Stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor Gastrointestinal Stromal Tumors (GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational thoracic tumors Hairy cell disease, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Histocytosis (e.g., Langerhans cell), Hodgkin's lymphoma, hypopharyngeal carcinoma, intraocular melanoma tumors, islet cell tumors (e.g., pancreatic neuroendocrine tumors), Kaposi's sarcoma, kidney cancer (e.g., renal cells, Wilms' tumor, childhood renal tumors, etc.), Langerhans cell histiocytosis, laryngeal cancer, white blood cells Hematologic diseases (e.g., acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (C) LL), chronic myeloid leukemia (CML), hairy cell, etc.), lip and oral cancer, liver cancer (primary ), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), lymphoma Tumors (e.g., AIDS-related, Burkitt's, cutaneous T-cell, Hodgkin's, non-Hodgkin's, primary central nervous system) 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 cervical squamous cell carcinoma of unknown primary site, midline duct carcinoma with Nat gene, oral cancer, multiple Endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, bone Myelodysplastic / myeloproliferative neoplasms, myeloid leukemia (e.g., chronic myeloid leukemia (CML)), Diseases (e.g., acute (AML), etc.), myeloproliferative neoplasms (e.g., chronic), nasal and paranasal Nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer (O ral Cancer), Oral Cavity Cancer (e.g. , lip, etc.), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer (e.g., upper epithelial, germ cell tumors, low-grade tumors, etc.), pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors) ), papillary tumors, paranasal sinus tumors, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer , pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate Cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and urethral transitional cell carcinoma, retinoblastoma, thoracic Rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue tissue, uterus, etc.), Sézary syndrome, skin cancer (e.g., childhood melanoma, Merkel cell carcinoma) , non-melanoma, 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 kidney Pelvic cancer, urethral cancer, uterine cancer (e.g., endometrial cancer), uterine sarcoma, vaginal cancer, vulvar cancer , Waldenström's macroglobulinemia, Wilms' tumor, etc. Not limited to.

[0206] The treatment methods described herein may, in some cases, be administered after one or more prior conventional treatments. For example, in the case of oncology, the methods described herein can be performed on subjects undergoing In some cases, for example, conventional chemotherapy, conventional radiation therapy, conventional immunotherapy This may be performed following conventional cancer therapy, including, but not limited to, chemotherapy, surgery, etc. In some cases, the methods described herein are useful in treating a range of conditions, including: a) a subject who has not responded to conventional therapy or In some cases, the methods described herein may be used when the patient is refractory to treatment. may be used if the subject is responding to conventional therapy.

[0207] In some cases, the disclosed methods may be used to treat minimal residual disease (e.g., rheumatoid arthritis) remaining after prior cancer treatment. The present invention can be used to target, treat, or eliminate a subject for MRD. Targeting, treating, and / or eliminating MRD may be important, whether MRD is refractory to prior therapy or not. Regardless, the method may be pursued using the present disclosure. to determine whether MRD has occurred following prior therapy or treatment other than with a multispecific antibody as described herein. Subjects with MRD after determination of refractory to one or more available treatment options It may be targeted, treated, and / or eliminated.

[0208] In some cases, the methods may be used prophylactically for surveillance. For example, a subject in need thereof may be a patient in need thereof, where the subject does not have detectable disease but has, for example, drug tolerance. and administering the antibodies described herein to patients at risk of developing recurrent cancer, including advanced cancer. In some cases, a prophylactic approach may be administered. The subject is a patient who is predicted to be drug resistant or who is predicted to become drug resistant. It may be used when a person is at particularly high risk of developing a primary cancer. A preventative approach is one in which a subject has previously been treated for cancer and is currently undergoing treatment for recurrent or drug-resistant cancer. It can be used when there is a risk of development.

[0209] In some cases, the methods of the disclosure involve targeting the expression of one or more markers or therapeutic targets. For example, in some cases, the method may involve analyzing the cancer for Cancer samples from the study were analyzed to determine whether the cancer expressed ABCC1 above a predetermined threshold. This may involve determining whether

[0210] In some cases, whether a subject is treated with an antibody of the present disclosure depends on the ABC For example, in some cases, the cancer may be at a pre-determined threshold. If the subject expresses ABCC1 at or above 1000 kJ / mL, then the subject may be administered an anti-A If the cancer expresses ABCC1 below a predetermined threshold, it can be treated with a BCC1 antibody. The subject may then not be treated with the anti-ABCC1 antibodies of the present disclosure.

[0211] Any convenient assay can be used to analyze ABCC1 levels, for example: Flow cytometry, nucleic acid-based assays (e.g., amplification, sequencing, etc.), cell sampling Any convenient biomedical technique may be used, including but not limited to immunohistochemistry, immunocytochemistry, and the like. Body samples may be used, including but not limited to, cancer biopsy samples. Useful predetermined thresholds for assessing the expression of the above markers and / or targets can be determined by any convenient method. can be determined by any suitable method and comparing the measured expression levels with corresponding controls. For example, in some cases, useful information about the level of ABCC1 in a sample may be obtained. The predetermined threshold is based on the level of ABCC1 measured in a reference cell, such as a healthy / normal cell. We can accommodate this.

[0212] How to make it As summarized above, the methods of the present disclosure also include methods for producing antibodies as described herein. and / or methods for identifying the antibody of interest. They can be produced by conventional synthetic methods, recombinant DNA methods, and the like for synthesis.

[0213] If the subject antibody is a single polypeptide chain, it can be synthesized using standard chemical peptide synthesis techniques. When a polypeptide is chemically synthesized, the synthesis can be carried out in a liquid phase. Alternatively, the C-terminal amino acid of the sequence may be bound to an insoluble support, followed by solid phase binding. Solid phase polypeptide synthesis (SPPS), in which the remaining amino acids are added sequentially to the sequence, is used to Examples of suitable methods for the chemical synthesis of hydroxybenzoates include various forms of SPPS, such as Fmoc and Boc. are available for synthesizing antibodies of interest.

[0214] Standard recombinant methods can be used for the production of the subject antibodies. For example, any Nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are incorporated into expression vectors. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains can be The peptide is operably linked to a control sequence in the expression vector that ensures expression of the peptide. Regulatory sequences include promoters (e.g., naturally associated or heterologous promoters), signal sequences, and the like. These elements include, but are not limited to, transcriptional regulators, enhancer elements, and transcription termination sequences. The control sequence transforms or transfects eukaryotic host cells (e.g., COS or CHO cells). eukaryotic promoter systems in vectors that can be transfected Once the vector is integrated into a suitable host, the host will express high levels of the nucleotide sequence. They are maintained under conditions suitable for expression, and collection and purification of the antibodies.

[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 synthesized by de novo solid-phase DNA synthesis or by the synthesis of a desired polymerase. Polymerase chain reaction (PCR) mutations of variants prepared earlier than oligonucleotides Oligonucleotide-mediated mutagenesis can be performed by cleaving a target polypeptide. Examples of suitable methods for preparing substitution, deletion, and insertion variants of peptide DNA are given below. See Adelman et al., DNA 2:183 (1983). Simply stated, the target polypeptide DNA is an oligonucleotide encoding the desired mutation. The hybridization sequence is modified by hybridizing the hybrid with a single-stranded DNA template. After the insertion, DNA polymerase is used to incorporate the oligonucleotide primers. A second complementary strand of the template is synthesized in its entirety, encoding the selected modification in the target polypeptide DNA. To make.

[0216] Suitable expression vectors are typically expressed as episomes or as an integral part of the host chromosomal DNA. An essential part of an expression vector is that it is replicable in a host organism. A selectable marker (e.g., ampicillin) that allows for detection of those cells transformed with the sequence. Phosphate resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance It contains α-amycin resistance.

[0217] Escherichia coli clones the polynucleotide encoding the antibody of interest. Examples of prokaryotic host cells that can be used for scanning are: Microbial hosts include bacilli such as Bacillus subtilis, and Salmonella, Other Enterobacteriaceae include Serratia, and various Pseudomonas species.

[0218] Other microorganisms, such as yeast, are also useful for expression. erevisiae and Pichia are examples of suitable yeast host cells, and may be used to develop A suitable vector having the appropriate control sequences (e.g., promoter), origin of replication, termination sequences, etc. Typical promoters include those encoding 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, alcohol dehydrogenase, Promoted from cytochrome C and enzymes involved in maltose and galactose utilization This includes the target.

[0219] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) using the methods of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). The polypeptides of the present invention can also be expressed and produced by Winnacker, Fro m Genes to Clones, VCH Publishers, NY, N. See, Y. (1987). Suitable mammalian host cells include CHO cell lines, various C os cell lines, HeLa cells, HEK cells, myeloma cell lines, and transformed B cells or hybrid Expression vectors for these cells include expression control sequences, e.g., Origins, promoters, and enhancers (Queen et al., Immunol Rev. 89:49 (1986), as well as ribosome binding sites and RNA splice sites. Necessary processing information such as transcriptional sequences, polyadenylation sites, and transcription terminator sequences Examples of suitable expression control sequences include those derived from immunoglobulin genes, SV40, Promoter derived from adenovirus, bovine papillomavirus, cytomegalovirus, etc. Co et al., J. Immunol. 148:1149 (1992) ) for more information.

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

[0221] kit Aspects of the present disclosure also include kits. The kits may include, for example, antibodies described herein, It may include any combination of reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. The subject kits include one or more of the subject antibodies, nucleic acids encoding same, or cells containing nucleic acids of interest. The kits can be configured for a variety of purposes, for example, therapeutic kits. (For example, the kit may include an anti-ABCC1 antibody and one or more additional agents, such as, for example, chemotherapy.) (which may contain an active agent), a kit for producing an antibody, a kit for screening an antibody This includes things like .

[0222] Optional components of the kit may vary, e.g., buffers, protease inhibitors, etc. When the subject kit includes a subject nucleic acid, the nucleic acid may also contain restriction sites, multiple cloning sites, and The various components of the kit may be present in separate containers. or certain compatible components may be pre-assembled into a single container, if desired. It can be done.

[0223] In addition to the above components, the subject kits may also include other kit components for practicing the subject methods. The instructions for practicing the subject methods may generally include instructions for using the subject methods. For example, the instructions may be recorded on a substrate such as paper or plastic. The instructions may therefore be printed on the container of the kit or its components as a package insert. may be present on the label (i.e., associated with the packaging or subpackaging) In other embodiments, the instructions are stored on a suitable computer-readable storage medium, e.g. , Compact Disc Read-Only Memory (CD-ROM), Digital Versatile Disc It exists as an electronic storage data file that resides on a DVD, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but are provided from a remote source. Means for obtaining the instructions are provided, for example, via the internet. Examples of forms include a web address where the instructions can be displayed and / or a link where the instructions can be downloaded. The kit includes a web address for obtaining the instructions, as well as the means for obtaining the instructions. is recorded on a suitable substrate. [Example]

[0224] The following examples provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. It is presented for the purpose of illustration only and is not intended to limit the scope of what the inventors regard as their invention. The experiments below are not illustrated and are not 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.). Although experimental results have been obtained, some experimental error and deviation should be considered. Unless otherwise specified, percentages are percentages by weight, molecular weights are weight average molecular weights, and temperatures are in degrees Celsius. and the pressure is atmospheric or near atmospheric.

[0225] Common methods in molecular and cellular biochemistry are Molecular Cloning ng:A Laboratory Manual,3rd Ed.(Sambrook et al., HaRBor Laboratory Press 2001), Sho rt Protocols in Molecular Biology,4th Ed (Ausubel et al. eds., John Wiley & Sons 1 999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy(Wagner et al. eds.,Academic Press 1999), Viral Vectors (Kaplift & Loew y eds., Academic Press 1995), Immunology M ethos Manual(I. Lefkovits ed.,Academic P ress 1997), and Cell and Tissue Culture:Lab oratory Procedures in Biotechnology(Doyl e & Griffiths, John Wiley & Sons 1998) found in standard textbooks, the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells for the methods mentioned or related to this disclosure Cells and kits were purchased from BioRad, Agilent Technologies, and The rmo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc. and commercial vendors such as, for example, Addgene, Inc., American Obtained from repositories such as the American Type Culture Collection (ATCC) It is possible to obtain it.

[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 Spleen and lymph node cells from vaccinated animals were used as SP The hybridoma supernatant was fused with 2 / 0 myeloma cells (hybridoma technology). Cells were screened for the presence of anti-ABCC1 antibodies by cytometry. CDRs from mouse IgG were transfected using standard protocols as described below. Expression of full-length IgG1 antibodies in HEK293 host cells via transfection and For production, it was cloned into a mammalian IgG1 backbone expression vector.

[0227] Expression vector For the generation of antibody expression vectors, the variable regions of the heavy and light chain DNA sequences are derived from human IgG Either the human IgG1 constant heavy chain or the human IgG1 kappa constant light chain was expressed in mammalian cell lines. The fragments were inserted into the optimized general recipient expression vectors and used for gene expression. The expressed genes were subcloned into the full-length genomic DNA for high-level gene expression. pCI-neoHuman, which uses the human cytomegalovirus (CMV) immediate early promoter The two antibody chains were cloned into two different mammalian expression vectors (Promega). The vector was cloned into

[0228] The N-terminal signal sequences from mouse IgG heavy chain and kappa light chain were added to the heavy and light chains, respectively. The signal peptide was cleaved during expression, leaving an intact N-terminal In the Fab construct, the C-terminus of the CH1 IgG1 constant region was left uncut for purification. was fused to a 6xHis tag.

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

[0230] Approximately 6 days after transfection, cells were harvested by centrifugation. 1 μg of total coding DNA per 1 transfected culture was added to Opti-MEM ( Exp. 100 mg / mL (Life Technologies) medium (registered trademark) and diluted in the same medium. Immunofluorescence assay with IFECTAMINE reagent (Life Technologies) for 20 min. The mixture was then added to Expi293® Expression Medium (Life Expi293® cells grown in suspension in PBS (Bio-Rad Technologies) 2.5 million cells / ml at 37°C with and covered with 8% CO2 in air. After 6 days, the medium containing the antibody constructs was collected by centrifugation.

[0231] mAb purification To purify antibody formats containing human Fc, add 10 μl per ml of supernatant. MabSelect™ SuRe™ (GE Healthcare) The Protein A resin was added to the medium and stirred overnight at 4°C. The next day, the Protein A resin was transferred to a vacuum manifold. A 24-well plate was prepared using a plate unit (Pall Lifesciences, USA). The resin was washed with PBS and the antibody was applied to a filter plate. Eluted at 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 preparation are as described by the manufacturer. Protein Express LabChip Kit (Perkin-E Caliper's LabChip GXII high-throughput The chips were placed in a buffer containing 0.2% SDS and a fluorescent dye. The polymer solution was automatically primed on the instrument. The destaining channel was primed with SDS and The polymer solution was filled with a dye-free polymer solution with or without DDT. Regardless of the sample size, reduction can be achieved by mixing a small amount of sample (2-5 μL) with caliper sample buffer. Proteins under original and non-reducing conditions were prepared. Samples were denatured at 75°C for 5 minutes. The mixture was centrifuged at 2000 g for 3 minutes and then run. Generated by GXII Touch software (Perkin Elmer).

[0233] Analytical Testing for mAb (HPLC) The purity and monomer content of the final protein preparation were assessed by high-throughput HPLC. Size exclusion chromatography (SEC) was performed using Infinite Advancebio SEC on a y1260 Agilent HPLC system 300A 4.6x300mm, 2.7um(p / n PL1580-5301)(Ag The assay was performed using a PBS solution containing 400 mM NaCl. Injections were performed under isocratic elution conditions using a mobile phase of sodium, pH 7.4, and the concentration was measured at 280 nm. Quantification was based on the relative areas of the detected peaks.

[0234] The subject antibodies are 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 cell debris, macromolecules other than the antibody of interest, etc. stomach.

[0235] Monoclonal antibody titration binding to KPC1 The binding titration of the recombinant antibody to KPC1 transfectants was performed at approximately 666 nM of antibody. The diluted antibodies in flow cytometry buffer were mixed with the cells and serially diluted from 0.1% to 1.0%. Both were incubated on ice for 30 minutes. Then, they were washed twice with flow cytometry buffer. Afterwards, the bound antibody was analyzed by immunoblotting using PE-labeled F (ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) and incubated with cells on ice for 20 minutes. After washing twice with buffer, fluorescent analysis was performed on an Attune NxT flow cytometer. Light was measured. Data were analyzed using GraphPad Prism 8.0 software. The EC50 was determined.

[0236] Cell binding assay Antibody binding to cells was assessed by flow cytometry. 293T cells stably transfected to express BCC1 were analyzed by flow cytometry. Wash once in filtration buffer (PBS + 2% FBS + 0.02% sodium azide) The cells were then resuspended in flow cytometry buffer at 2 x 10^6 cells / mL and analyzed in 96-well plates. The recombinant antibody was added to a microtiter plate at 0.1 mL / well. Add to cells at 5µg / mL for consistency or dilute to 1µg / mL in flow cytometry buffer. The cells were serially diluted from 00 μg / mL. After incubating the cells on ice for 30 minutes, After washing twice with flow cytometry buffer, the bound antibody was purified by PE-labeled F(ab')2 Detected by fragmented goat anti-human IgG (Jackson ImmunoResearch) The EC50 was calculated as the maximum response. It is calculated to be the concentration of antibody that gives half the answer.

[0237] ABCC1 efflux assay HEK293T cells expressing human ABCC1 were washed several times and incubated in phenol red-free 1 x 10 per ml in DMEM 6 Aliquots of 50 μl at a cell density of 100 cells / well The cells were mixed with 50 μl of antibody and aliquoted into a 96-well plate. The cells were then incubated for 1.5 hours at 7°C. The cells were then washed twice and finally The cells were resuspended in 1 ml of PBS. Calcein AM fluorescence was measured by flow cytometry. Ta.

[0238] Cytotoxicity assay The effect of anti-ABCC1 antibodies on vincristine cytotoxicity was investigated in ABCC1-expressing mice. The cells were plated in white flat-bottom plates. In a 96-well tissue culture plate, add 0.05 mL of assay medium (DMEM + 10% FBS ) at 5000 cells / well. Vincristine was added at 100 μg / mL (2× The assay medium containing the test or control antibody at a final concentration of 200 μM was Prepared at 2x final assay concentration by dilution. Add an equal volume ( 0.05 mL) was added to 293T_ABCC1 cells in a 96-well plate. The plates were then incubated at 37°C in 5% CO2. After approximately 72-96 hours, The cells were equilibrated to room temperature and then filled with Promega® CellTiter-Glo®. Cell viability was assessed using a luminescent cell viability assay according to the manufacturer's recommended protocol. Luminescence was measured using a Molecular Devices® FlexSta tion®3 multimode microplate reader and GraphP Analysis was performed using Prism 8.0 software. Half-maximal inhibitory concentration (IC50) is a drug (vincristine or other chemotherapy cytotoxic agent) that reduces response (cell growth) by 50%. The concentration of the inhibitor.

[0239] CT26 syngeneic xenograft mouse model Syngeneic models are allografts immortalized from mouse cancer cell lines and then cultured in the same manner. CT26 is a BAL mouse strain with invasive colon cancer. N-nitroso-N-methylurethane (NNMU)-induced immunization established from B / c mice C26 cells are a differentiated fibroblastic colon cancer cell line. 2638™) and was supplemented with 10% FBS, 1% penicillin, 1% streptomycin, and They were maintained in RPMI-1640 medium supplemented with tomycin at 37°C and 5% CO2. The cell line used was confirmed to be authentic and mycoplasma negative. The cells adhered to the blast morphology and developed tumors after transplantation into syngeneic BALB / c mice or immunodeficient mice. It forms tumors and metastases.

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

[0241] Tumor 100-150mm 3 Once the mice reached 100 mg / kg, they were randomly divided into six groups of five mice each. It was artificialized. 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. The antibody was administered 4 hours before doxorubicin.

[0243] Tumors were measured using calibrated calipers and tumor volumes calculated according to the formula 1 / 2*L*S*S. Measured three times a week using the formula, where L is the long axis and S is the short axis of the tumor. Body weights were recorded and continuously monitored throughout the study. In addition to the above predefined criteria, rapid weight loss, loss of ability to walk, difficulty breathing, or inability to drink water Animals were euthanized when they became moribund due to inability to eat.

[0244] result Table 3 lists the following characteristics of anti-ABCC1 antibodies: Human as measured by FACS Binding to 293T cells stably transfected to express ABCC1 and The cells were stably transfected to express cynomolgus monkey ABCC1 as measured by FACS. Binding to infected 293T cells. [Table 3]

[0245] Figure 1 shows the results of the flow cytometry (FACS) assay using ABCC1. The doxorubicin-resistant lung cancer cell line H69AR (ATCC® CR) expresses Titration results of 10 anti-ABCC1 monoclonal antibodies against L-11351™ All antibodies tested showed significant (more than 10-fold) backbone activation in H69AR cells. Ground fluorescence mean intensity (FMI) binding is shown.

[0246] 2A-2B show the results of human and cynomolgus monkey ABCC1 in the above FACS assay. Anti-ABCC1 monoclonal antibody shown against overexpressing rat C6 glioma cell line All antibodies tested bind to C6 cells overexpressing human ABCC1. The antibody showed significant binding to both C6 cells overexpressing cynomolgus monkey ABCC1 and C6 cells overexpressing cynomolgus monkey ABCC1. show.

[0247] 3A to 3C, 4, 5A to 5B, and 6A to 6B show the results of the FACS assay described above. Further studies on human and cynomolgus monkey ABCC1-overexpressing rat C6 glioma cell lines The results of titration binding of anti-ABCC1 monoclonal antibodies are shown. "Second Ab only" is negative. Refers to a non-primary antibody used as a control. All anti-ABCC1 antibodies tested showed negative results. Compared with controls, C6 cells overexpressing human ABCC1 and C6 cells overexpressing cynomolgus monkey ABCC1 were The antibody showed significant binding to both C6 cells overexpressing IgG1 and C6 cells.

[0248] Figures 7A-7B show a comparison of the results of a 2D ELISA performed using HEK293T cells expressing human ABCC1. Results of a BCC1 shedding assay are shown. All anti-ABCC1 antibodies tested were It significantly inhibits the efflux function of transporters.

[0249] Figures 8A-8C show titration binding and efflux assays of humanized anti-ABCC1 monoclonal antibodies. We present the characterization of humanized anti-ABCC1 antibodies C1.831.hu11 and C1.861. .hu11, C1.861.hu21, and C1.844.hu21 were titration binding assays. Binding to both human and cynomolgus monkey ABCC1 in B1 and B2 in efflux assays Significantly inhibits the efflux function of the CC1 transporter.

[0250] 9A to 9C show the results of the rat C6 glioma cell line overexpressing human and cynomolgus monkey ABCC1. Anti-ABCC1 monoclonal antibodies C1.831, C1.861, and C1.84 Figure 4 shows the binding of humanized variants of 4. All humanized antibodies bind to human and cynomolgus ABC It retains the ability to bind both C1 and C2.

[0251] 10A-10B show the results of human and cynomolgus monkey ABCC1-overexpressing rat C6 glioma cells. Humanized anti-ABCC1 antibodies C1.851.12, C1.851.14 and C1.851.15 against cell lines All three antibodies show binding to human and cynomolgus monkey ABCC1. It retains the ability to bind to both

[0252] 11A-11C show human and crab overexpressing ABCC1 and KT9, respectively. Binding of four humanized ABCC1 / KT9 bispecific antibodies to the monkey C6 cell line A schematic bispecific antibody structure is also shown. KT9 is an anti-PD-L1 monoclonal antibody. The monoclonal antibody atezolizumab is shown. Bispecific antibodies include the ABCC1 antibody shown. The heavy and light chains from the KT9 antibody and the scFv region formed from the KT9 antibody are included. All bispecific antibodies tested were expressed in C6 cells overexpressing ABCC1 and KT9 cells overexpressing ABCC1. It binds to both C6 cells.

[0253] 12A to 12C show the results of 293T cells expressing human ABCC1 and human or Four humanized C1 / KT1 dual-antibody clones were tested against 293T cells expressing cynomolgus monkey KT1. KT1 is an anti-ErbB2 (anti-HER2) monoclonal antibody. The bispecific antibody is trastuzumab, which is a fusion protein derived from the anti-ABCC1 antibody shown. The heavy and light chains and the scFv region formed from the KT1 antibody were included. The bispecific antibody was synthesized by 293T cells expressing human KT1 and cynomolgus monkey KT1. Ability to bind both to 293T cells and to 293T cells expressing human ABCC1 Hold.

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

[0255] Figure 16 shows that the three anti-ABCC1 monoclonal antibodies tested inhibited H69AR cytotoxicity. The assay was performed in human small cell lung cancer cells (HCC) and showed that the compound inhibited tumor growth in vivo in a human small cell lung cancer cell line (HCC). H6, an adriamycin-selected C1-positive variant of the lung cancer cell line, NCI-H69 The effect of the tested antibodies on vincristine cytotoxicity to the 9AR cell line is evaluated.

[0256] Figure 17 shows the effect of anti-ABCC1 monoclonal antibodies C1-831 and C1-737A on CT 26 and demonstrate inhibition of tumor growth in vivo in a syngeneic mouse tumor model.

[0257] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding. However, certain changes and modifications may be made without departing from the spirit or scope of the appended claims. It will be readily apparent to those skilled in the art in light of the teachings of the present invention that adjustments can be made.

[0258] Accordingly, the foregoing merely illustrates the principles of the present invention. Although not described or illustrated in the specification, it embodies the principles of the present invention and is included within its spirit and scope. It is understood that various configurations can be devised. All examples and conditional language have been provided by the inventors primarily in furtherance of the principles and techniques of the present invention. It is intended to assist the reader in understanding the concepts contributed and The examples and conditions set forth should not be construed as limiting the scope of the present invention. All statements herein reciting aspects, aspects, and embodiments, and specific examples thereof, are intended to be illustrative and not restrictive of the structure thereof. In addition, such equivalents are not currently Both known equivalents and future-developed equivalents, i.e., equivalents that perform the same function, regardless of structure, It is intended to include any developed element that performs the functions disclosed herein. Any disclosure made in any way without regard to whether such disclosure is expressly recited in the claims is unrelated. However, it is not intended to be dedicated to the public.

[0259] The scope of the present invention, therefore, is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is defined by the appended claims. 112(f) or 1 U.S.C. Section 12(6) requires that the precise phrase "means for" be used at the beginning of such a limitation in a claim. or the exact phrase "steps for" is recited in the claims and such exact phrase is expressly defined as being incorporated for the purposes of the patent claim. 112(f) or 35 U.S.C. § 112(f) unless used to limit the scope of a claim. Article 112(6) will not be invoked.

Claims

1. ATP-binding cassette subfamily C member 1 (ABCC1) on the surface of mammalian cells an antibody that specifically binds to ABCC1, wherein the antibody specifically binds to Heavy chain phases of pairs of variable heavy (VH) and variable light (VL) chain regions of antibodies listed in Table 2 Complementarity determining regions 1-3 (HCDR1-3) and light chain CDR1-3 (LCDR1-3) Antibodies that compete with antibodies.

2. 1. The antibody of claim 1, wherein the antibody comprises HCDRs 1-3 of the VH region of an antibody listed in Table 2. The antibody described in

3. 2. The antibody of claim 1, wherein the antibody comprises LCDRs 1 to 3 of the VL region of an antibody listed in Table 2. The antibody described in

4. The antibody, Heavy chain phases of pairs of variable heavy (VH) and variable light (VL) chain regions of antibodies listed in Table 2 The antibody of claim 1 , comprising a complementarity determining region (HCDR) and a light chain CDR (LCDR).

5. ATP-binding cassette subfamily C member 1 (ABCC1) on the surface of mammalian cells An antibody molecule that specifically binds to

6. The antibody (a) Heavy chain complementarity determining regions 1-3 (HCDR1-3) of the VH region of an antibody listed in Table 2 a variable heavy (VH) region comprising: (b) comprising light chain CDRs 1-3 (LCDRs 1-3) of the VL region of an antibody listed in Table 2 a variable light (VL) region, (c) a VH region comprising HCDRs 1-3 of the VH region of an antibody listed in Table 2, and a VL region comprising LCDR1-3 of the VL region of the listed antibody; or (d) a VH region comprising HCDRs 1 to 3 of the VH region of an antibody listed in Table 2, and 6. The antibody molecule of claim 5, comprising a VL region comprising LCDRs 1 to 3 of the VL region of the antibody.

7. The antibody comprises HCDRs 1-3 and HCDRs 1-3 of a pair of VH and VL regions of an antibody listed in Table 2. The antibody molecule of claim 6, comprising LCDR1-3.

8. The antibody comprises HCDRs 1-3 of the VH region of a first antibody listed in Table 2.

6. The antibody described in 6.

9. 9. The antibody of claim 8, wherein the antibody comprises LCDRS1 to LCDRS3 of the VL region of a second antibody in Table 2. The antibody molecule described in

10. The antibody molecule comprises the variable light (VL) chain and / or variable heavy (VH) chain of an antibody listed in Table 2. The antibody molecule of claim 6, comprising a chain.

11. The antibody i. C1.831.hu41 antibody, C1.831.hu11 antibody, C1.831.hu21 antibody, C1.831.hu31 antibody, C1.831.hu41 antibody, C1.831.hu1 ...11 antibody, C1.831.hu41 antibody, C1.83 1.

844. hu21 antibody, C1.

851. hu15 antibody, C1.

851. hu12 antibody , or the heavy chain complementarity determining regions 1 to 3 (HCDs) of the VH region of the C1.861.hu11 antibody a variable heavy chain (VH) region comprising: ii. C1.831.hu41 antibody, C1.831.hu11 antibody listed in Table 2; C1.

844. hu21 antibody, C1.

851. hu15 antibody, C1.

851. hu12 anti or the light chain CDRs 1 to 3 (LCDR1 to 3) of the VL region of the C1.861.hu11 antibody. 3), a variable light chain (VL) region comprising iii. C1.831.hu41 antibody, C1.831.hu11 antibody listed in Table 2 , C1.

844. hu21 antibody, C1.

851. hu15 antibody, C1.

851. hu12 antibody, or a VH region comprising HCDR1-3 of the VH region of the C1.861.hu11 antibody , and the C1.831.hu41 antibody, C1.831.hu11 antibody, C1.831.hu21 antibody, C1.831.hu31 antibody, C1.831.hu41 antibody, C1.831.hu1 ...21 antibody, C1.831.hu31 antibody, C1.831.hu41 antibody, C1. 1.

844. hu21 antibody, C1.

851. hu15 antibody, C1.

851. hu12 antibody or a VL region comprising LCDR1-3 of the VL region of the C1.861.hu11 antibody; iv. C1.831.hu41 antibody, ... 11 antibody, C1.

844. hu21 antibody, C1.

851. hu15 antibody, C1.

851. HCD of the VH and VL regions of the hu12 antibody or C1.861.hu11 antibody a VH domain comprising R1-3 and a VL domain comprising LCDR1-3; or v. C1.831.hu41 antibody, C1.831.hu11 antibody, C1.831.hu21 antibody, C1.831.hu31 antibody, C1.831.hu41 antibody, C1.831.hu1 ...11 antibody, C1.831.hu41 antibody, C1.83 1.

844. hu21 antibody, C1.

851. hu15 antibody, C1.

851. hu12 antibody or the VH and VL regions of the C1.861.hu11 antibody. antibody molecule.

12. The antibody i. C1.830B.hu11 antibody, C1.851.hu11 antibody listed in Table 2; C1.

851. hu13 antibody, C1.787a. hul1 antibody, C1.

844. hu11 Antibody, C1.

861. hu21 antibody, C1.

861. hu41 antibody, C1.

861. hu VH comprising HCDR1-3 of the VH region of antibody C1.61 or C1.851.hu14 region, ii. C1.830B.hu11 antibody, C1.851.hu11 antibody listed in Table 2 , C1.

851. hu13 antibody, C1.787a. hul1 antibody, C1.

844. hu1 1 antibody, C1.

861. hu21 antibody, C1.

861. hu41 antibody, C1.

861. h VL region comprising LCDR1-3 of the u61 antibody or C1.851.hu14 antibody L area, iii. C1.830B.hu11 antibody, C1.851.hu11 antibody listed in Table 2 Body, C1.

851. hu13 antibody, C1.787a. hul1 antibody, C1.

844. hu 11 antibody, C1.

861. hu21 antibody, C1.

861. hu41 antibody, C1.

861. Contains HCDR1-3 of the VH region of the hu61 antibody or C1.851.hu14 antibody VH regions, and the C1.830B.hu11 antibodies, C1.851.hu1 listed in Table 2 1 antibody, C1.

851. hu13 antibody, C1.787a. hul1 antibody, C1.

844. hul1 antibody, C1.

861. hu21 antibody, C1.

861. hu41 antibody, C1.86 1. LCDR1 to LCDR3 of the VL region of the hu61 antibody or C1.851.hu14 antibody a VL region comprising iv. C1.830B.hu11 antibody, C1.851.h, respectively, listed in Table 2 u11 antibody, C1.

851. hu13 antibody, C1.787a. hu11 antibody, C1.84 4. hul1 antibody, C1.

861. hu21 antibody, C1.

861. hu41 antibody, C1. VH and VL regions of the 861.hu61 antibody or the C1.851.hu14 antibody a VH region comprising HCDR1-3 and a VL region comprising LCDR1-3 of v. C1.830B.hu11 antibody, C1.851.hu11 antibody listed in Table 2; C1.

851. hu13 antibody, C1.787a. hul1 antibody, C1.

844. hu11 Antibody, C1.

861. hu21 antibody, C1.

861. hu41 antibody, C1.

861. hu 61 antibody, or C1.851.hu14 antibody.

5. An antibody molecule according to claim 5.

13. The antibody i. C1.773 antibody, C1.773a antibody, C1.777a antibody listed in Table 2; C1.784a antibody, C1.786a antibody, C1.787a antibody, C1.827 antibody, C 1.830B antibody, C1.831 antibody, C1.835 antibody, C1.841 antibody, C1.8 44 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 represents a VH region comprising HCDR1-3 of the VH region of the C1.879A antibody; ii. C1.773 antibody, C1.773a antibody, C1.777a antibody listed in Table 2 , 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 anti antibody, C1.861 antibody, C1.863 antibody, C1.876 antibody, C1.877 antibody, or a VL region comprising LCDR1 to LCDR3 of the VL region of the C1.879A antibody; iii. C1.773 antibody, C1.773a antibody, C1.777a antibody listed in Table 2 body, 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, young or the VH region comprising HCDRs 1-3 of the VH region of the C1.879A antibody, and the VH regions of the VH regions of the C1.879A antibody and ... C1.773 antibody, C1.773a antibody, C1.777a antibody, C1.784a antibody body, 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 anti antibody, C1.863 antibody, C1.876 antibody, C1.877 antibody, or C1.879A a VL region comprising LCDR1-3 of the VL region of an antibody; iv. C1.773 antibody, 773a antibody, 777a antibody, respectively, listed in Table 2; 784a antibody, 786a antibody, 787a antibody, C1.827 antibody, C1.830B antibody, C1.831 antibody, C1.835 antibody, C1.841 antibody, C1.844 antibody, C1.8 45 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 The VH region and the VL region each contain HCDRs 1-3 and LCDRs 1-3, respectively. a VL region, or v. C1. 773 antibody, 773a antibody, 777a antibody, 784a antibody listed in Table 2 , 786a antibody, 787a antibody, C1.827 antibody, C1.830B antibody, C1.831 antibody, C1.835 antibody, C1.841 antibody, C1.844 antibody, C1.845 antibody, C 1.847 antibody, C1.851 antibody, C1.855 antibody, C1.861 antibody, C1.86 VH region of antibody C1.3, antibody C1.876, antibody C1.877, or antibody C1.879A and a VL region.

14. The antibody of any one of claims 11 to 13, wherein the antibody comprises a human IgG Fc region. antibody molecule.

15. The antibody of any one of claims 11 to 13, wherein the antibody comprises a human IgG1 Fc region. antibody molecule.

16. 14. The antibody of claim 11, wherein the antibody comprises a human IgG1 constant heavy chain and a constant light chain region. The antibody molecule of any one of claims 1 to 4.

17. When the antibody binds to cells expressing ABCC1, it inhibits the efflux of the cells by ABCC1.

10. An antibody molecule according to any one of the preceding claims which inhibits

18. 10. The antibody molecule of claim 1, wherein the antibody comprises a humanized light chain.

19. 10. The antibody molecule of claim 1, wherein the antibody comprises a humanized heavy chain.

20. The antibody may be a bispecific antibody, an Ig monomer, a Fab fragment, an F(ab') ２ Fragment, Fd The method of the preceding claims, wherein the antibody is selected from the group consisting of a fragment, scFv, scAb, dAb, and Fv. The antibody molecule of any one of claims 1 to 4.

21. The antibody is a double antibody comprising the VH and VL regions of any one of claims 1 to 20. a second specific antibody comprising HCDR1-3 of an antibody that binds to a tumor-associated antigen (TAA); 10. The antibody molecule of claim 1, further comprising a VH region of:

22. the antibody comprises a second VL region, and the second VH region and the second VL region are The antibody molecule of claim 21 which binds to the TAA.

23. the second VH region and the second VL region are present in a single polypeptide.

23. The antibody molecule of claim 22.

24. 23. The method of claim 22, wherein the second VH region and the second VL region are present in an scFv. The antibody molecule described.

25. The antibodies comprise a common light chain, the common light chain comprising a nucleotide sequence according to any one of claims 1 to 20.

22. The antibody molecule of claim 21, comprising a VL region as described above.

26. The antibody molecule of any one of claims 21 to 25, wherein the TAA is PD-L1. 。

27. The antibody molecule of claim 26, wherein the antibody that binds to PD-L1 is atezolizumab. child.

28. The bispecific antibody is selected from the group consisting of the C1.844 antibody and the C1.85 antibody, respectively, listed in Table 2. The VH region and LCDR1-3 of the VH region and VL region of antibody 1 are and a VL region comprising HCDR1-3 and LCDR1-3 of atezolizumab.

28. The antibody molecule of claim 27, comprising:

29. The bispecific antibody is a C1.844hu21 antibody or a C1.851 antibody listed in Table 2. VH and VL regions of the hu12 antibody and HCDR1-3 and LCDR of atezolizumab and scFvs comprising 1 to 3.

30. The TAA ErbB2 (HER2), an antibody according to any one of claims 21 to 25. molecule.

31. The antibody molecule of claim 30, wherein the antibody that binds to ErbB2 is trastuzumab. child.

32. The bispecific antibodies are the C1.844 antibody, C1.831 antibody, and the C1.842 antibody, respectively, listed in Table 2. or a VH region comprising HCDR1-3 of the VH region and VL region of the C1.851 antibody. and a VL region including LCDR1-3, and HCDR1-3 and LCDR1 of trastuzumab and an scFv comprising from 1 to 3.

33. The bispecific antibody is selected from the group consisting of C1.844hu21 antibody, C1.831h21 antibody, and C1.844hu21 antibody. The VH and VL regions of the u11 antibody or the C1.851hu12 antibody and trastuzumab and an scFv comprising HCDR1-3 and LCDR1-3 of body molecules.

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

34. An antibody molecule according to any one of claims 1 to 33.

35. 10. The antibody of claim 1, wherein the antibody comprises a VL region and a VH region present in a single polypeptide.

34. An antibody molecule according to any one of claims 1 to 33.

36. A method for treating cancer in a subject, comprising administering to a subject a composition comprising the compound of any one of claims 1 to 35. The antibody molecule of claim 1, wherein the method comprises administering the antibody to the subject. Antibody molecules for

37. The method further comprises administering the antibody in combination with at least one additional active agent. wherein the at least one additional active agent is a chemotherapeutic agent, a multidrug resistance transporter 37. The method of claim 36, comprising administering to a subject a therapeutic agent selected from the group consisting of an inhibitor of HIV-1, an immunotherapeutic agent, or a combination thereof. antibody molecule.

38. The at least one additional active agent is a chemotherapeutic agent, and optionally, the chemotherapeutic agent Drugs include taxol, vinca alkaloids, anthracyclines, etoposide, and mitoxin.

38. The antibody molecule for use according to claim 37, which is thiamin, thiamin, or methotrexate.

39. the subject being treated has been determined to be resistant to treatment with the chemotherapeutic agent; The antibody molecule for use according to any one of claims 36 to 38, wherein the patient has cancer.

40. 1. A pharmaceutical composition comprising: An antibody according to any one of the preceding claims; and a pharmaceutically acceptable excipient.

41. 41. The pharmaceutical composition of claim 40, further comprising an additional active agent.

42. 42. The pharmaceutical composition of claim 41, wherein the additional active agent is a chemotherapeutic agent.

43. 42. The method of claim 41 , wherein the additional active agent comprises an inhibitor of a multidrug resistance transporter. Pharmaceutical compositions.

44. 42. The pharmaceutical composition of claim 41, wherein the additional active agent comprises an immunotherapeutic agent.

45. 36. The antibody molecule of claim 1, wherein the antibody molecule comprises one or more sequences encoding the antibody molecule of claim 1. One or more nucleic acids.

46. 46. ​​One or more recombinant expression vectors comprising one or more nucleic acids of claim 45.

47. 47. Genetically modified with one or more recombinant expression vectors of claim 46. host cell.

48. A protein comprising an ABCC1-binding domain, a transmembrane domain, and an intracellular signaling domain. an immune effector cell comprising a CAR, the ABCC1-binding The domains are selected from heavy chain complementarity determining regions 1-3 of the variable heavy chain (VH) region of the antibodies listed in Table 2. (HCDR1-3), and / or the light chain C of the variable light chain (VL) region of the antibodies listed in Table 2 Immune effector cells comprising DR1-3 (LCDR1-3).

49. A method for assaying expression of ABCC1 on the cell surface of a cell, comprising:

36. A method comprising contacting a subject with the antibody of any one of claims 1 to 35.

50. 50. The method of claim 49, wherein the antibody is detectably labeled.

51. A method for inhibiting the efflux activity of ABCC1 expressed by a living cell, comprising: A method comprising contacting with an antibody according to any one of claims 1 to 35.

52. 52. The method of claim 51, further comprising contacting the cell with an inhibitor of ABCC1-mediated efflux. The method described below.

53. 53. The method of claim 51 or 52, further comprising contacting the cells with a chemotherapeutic agent. Law.

54. The method of any one of claims 51 to 53, wherein the cells are cancer cells.

55. 55. The method of claim 54, wherein the cancer cells are multidrug resistant cancer cells.

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