Anti-MDR1 antibodies and uses thereof

Abstract

Reagents that can be used to assay efflux pump (EP) expression and / or inhibit EPs are provided. [Solution] Antibodies that target the cellular efflux pump multidrug resistance protein 1 (MDR1) are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of MDR1 expression, the level of MDR1 expression, and / or inhibit MDR1 function in cells, such as tumor cells, are also disclosed. Additionally, multispecific antibodies that bind to MDR1 and tumor-associated antigens are provided. Also provided are methods of treating a subject for cancer, comprising administering to the subject an anti-MDR1 antibody disclosed herein, or a multispecific antibody that targets both MDR1 and a tumor-associated antigen.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 967,470, filed January 29, 2020, and U.S. Provisional Patent Application No. 62 / 967,470, filed September 2020. This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 085,818, filed on the 30th. and US Pat. No. 6,229,699, which applications are incorporated herein by reference in their entireties.

[0002] Incorporation by reference of sequence listing provided as a text file "KNJY-003WO SEQ LIST_ST25.txt" created on January 28, 2021, with a size of 291 KB The sequence listing is provided here as a text file: The contents of which are incorporated herein by reference in their entirety.

[0003] Drug resistance is a well-known phenomenon that occurs when a disease becomes resistant to drug treatment, and is commonly seen in tumors. Cancer is a major and growing problem in various fields of medicine, including cancer biology. Initially sensitive to chemotherapy, but over time, the drug becomes more susceptible to inhibition, degradation, and excretion. Resistance through these and other mechanisms, including DNA mutations and metabolic changes that promote potentiation can be developed. [Background technology]

[0004] Efflux pumps (EPs) are proteins expressed in living cells that transport various proteins from cells. ATP-binding cassette (ABC) transporters have evolved to naturally excrete these compounds. Members of the transporter family of proteins are examples of EPs that allow drug efflux. Although the structure of each protein varies (e.g., the human ABC family has 49 known members), (barred), all of which contain a highly conserved nucleotide-binding domain and a more variable They are classified by the presence of two distinct domains: a membrane-spanning domain with ATP binding activity. Multidrug resistance proteins encoded by the ABCB1 gene Among these, multidrug resistance protein 1 (MDR1) was the first to be identified. MDR1 expression is a key factor in the response to treatment with certain chemotherapy drugs and has been widely studied. Increase.

[0005] EP allows tumors to develop resistance to chemotherapy drugs. Such resistance is This chemotherapy resistance is often associated with enhanced efflux of chemotherapy drugs from drug-resistant cells. When applied to multiple chemotherapy drugs, it is called multidrug resistance (MDR). Summary of the Invention

[0006] Therefore, we develop reagents that can be used to assay for EP expression and / or inhibit EP. It is necessary.

[0007] Antibodies that target the cellular efflux pump MDR1 are provided. Also provided are pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits encoding the compounds. Detecting the presence or absence of MDR1 expression in cells (e.g., tumor cells) and the level of MDR1 expression. Also disclosed are methods of using the antibodies to inhibit MDR1 function and / or inhibit MDR1 function. Furthermore, multispecific mAbs that bind to MDR1 and tumor-associated antigens (TAA) Anti-MDR1 antibodies or antibodies targeting both MDR1 and a TAA are provided. Also provided are methods of treating a subject for cancer, comprising administering to the subject a multispecific antibody that can be. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows a chromatogram for the purified anti-MDR1 antibody of the present disclosure. A 92.08% pure IgG antibody (MW 171.74 kDa) was obtained. Traces of heavy / light dimers "HL" (MW 94.88 kDa) and heavy / heavy "HH" (MW 126.85 kDa) were also present. [Figure 2] 2A-2B show single-point binding of anti-MDR1 antibodies to 293T cells overexpressing MDR1. [Figure 3] 3A-3D show titration of anti-MDR1 monoclonal antibodies binding to MDR1. [Figure 4] 4A to 4D show the effect of the listed anti-MDR1 antibodies on vincristine (Vin) cytotoxicity. [Figure 5] 5A-5C show the efficacy of the listed anti-MDR1 antibodies against a drug-resistant ovarian cancer (A2780 ADR) model in nude mice. [Figure 6] FIG. 6 shows the efficacy of the listed anti-MDR1 antibodies against the drug-resistant MES-SA-DX5 uterine sarcoma model in nude mice. [Figure 7] FIG. 7 shows the efficacy of anti-MDR1 antibody B1.223 against the drug-resistant MES-SA-DX5 uterine sarcoma model in NSG mice. [Figure 8] FIG. 8 shows that ABCB1 knockout in drug-resistant tumors induces chemosensitivity to paclitaxel. [Figure 9] FIG. 9 summarizes the binding of the listed antibodies to various regions of human MDR1. [Figure 10]Figures 10A-10C show the binding of the B1.129 antibody and its humanized versions to human and cynomolgus monkey MDR1. [Figure 11] FIG. 11 shows the binding of the B1.28 antibody and its humanized versions to human and cynomolgus MDR1. [Figure 12] FIG. 12 shows the binding of the B1.261 antibody and its humanized versions to human and cynomolgus MDR1. [Figure 13] FIG. 13 summarizes the effect of the listed antibodies on the efflux of MDR1 substrates. [Figure 14] FIG. 14 shows the binding of anti-MDR1 and anti-CD47 bispecific antibodies to human MDR1 expressed on the surface of C6 cells. [Figure 15] FIG. 15 shows data on the binding of anti-MDR1 and anti-CD47 bispecific antibodies to cynomolgus monkey and human CD47 expressed on the surface of C6 cells. [Figure 16] FIG. 16 shows the binding selectivity titration of the listed bispecific antibodies against DX5 WT, DX5 B1 KO, and DX5 CD47 KO cell lines. [Figure 17] Figure 17A shows the binding of a humanized anti-MDR1 and anti-CD47 bispecific antibody to C6 cells overexpressing human ABCB1 (hB1), human CD47 (hKT14), or cynomolgus monkey CD47 (cKT14). Figure 17B shows the binding of a humanized anti-MDR1 and anti-CD47 bispecific antibody to C6 cells overexpressing human ABCB1 (hB1), human CD47 (hKT14), or cynomolgus monkey CD47 (cKT14). [Figure 18] Figures 18A-18B summarize the results of assaying various bispecific antibodies for binding to CD47 using ELISA or CD47-expressing cells and using ABCB1-expressing cells. ND = not determined. [Figure 19] FIG. 19 shows various bispecific antibody formats according to certain embodiments of the present disclosure. [Figure 20]FIG. 20 shows an anti-MDR1 and anti-CD47 bispecific antibody with scFv-Fab-Fc format. [Figure 21] FIG. 21 shows that the KT14.5F9hu scFv-B1.28.hu13 and KT14.B6H12hu scFv-B1.28.hu13 antibodies bind to both human CD47 and human MDR1 expressed on the surface of C6 cells. [Figure 22] FIG. 22 shows that the KT14.5F9hu scFv-B1.261.hu1 and KT14.B6H12hu scFv-B1.261.hu1 antibodies bind to both human CD47 and human MDR1 expressed on the surface of C6 cells. [Figure 23] 23A-23C show the effect of an anti-MDR1 and anti-CD47 bispecific antibody of the present disclosure when administered alone or co-administered with paclitaxel on tumor volume in a MES-SA-DX5 xenograft model. [Figure 24] FIG. 24 lists the various combinations of anti-MDR1 heavy chain, anti-HER2 heavy chain, and anti-MDR1 common light chain that were tested in a bispecific antibody format to identify combinations that bind to both antigens. [Figure 25] FIG. 25 lists the various combinations of anti-MDR1 heavy chain, anti-PDL1 heavy chain, and anti-MDR1 common light chain that were tested in a bispecific antibody format to identify combinations that bind to both antigens. DETAILED DESCRIPTION OF THE INVENTION

[0009] definition The terms "antibody" and "immunoglobulin" refer to antibodies of any isotype or or immunoglobulins, antibody fragments (Fab, Fv, scF) that retain specific binding to the antigen F(ab')2, chimeric antibodies, humanized antibodies, Monoclonal antibodies, single-chain antibodies (including antibodies containing only heavy chains (e.g., VHH camelid antibodies), Bispecific antibodies and fusion proteins comprising antigen-binding portions of antibodies and non-antibody proteins Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins are divided into different classes. There are five major types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. There are classes, some of which are further divided into subclasses (isotypes), e.g., IgG1, IgG2 , IgG3, IgG4, IgA, and IgA2. The terms "antibody" and "immunoglobulin" are used interchangeably. " specifically includes, but is not limited to, IgG1, IgG2, IgG3 and IgG4 antibodies. The antibodies are Detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, etc. The antibody can be bound to, for example, biotin (a member of the biotin-avidin specific binding pair). The antibody may be further conjugated to other moieties, such as members of a specific binding pair such as Also, solid supports, including but not limited to polystyrene plates or beads, may be used. The antibody may be monovalent or bivalent. The antibody may bind to a toxin such as a chemotherapeutic agent. The hydroxyl group may be conjugated to a hydroxyl group.

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

[0011] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region , a tight, non-covalent association of one heavy chain variable domain and one light chain variable domain. In this configuration, the three CDRs of each variable domain interact to form a VH-VL dimer. The six CDRs define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, the Fv, which contains only a single variable domain (or three CDRs specific for an antigen), Even half of a single CDR from each variable domain has a lower affinity than the complete binding site containing the three CDRs of each variable domain. Although they have a low level of activity, they have the ability to recognize and bind to antigens.

[0012] A "Fab" fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ) The Fab fragment also contains a few residues at the carboxyl terminus of the heavy chain CH1 domain. Fab' fragments by the addition of one or more cysteines from the antibody hinge region Fab'-SH is different from Fab'-SH, in which the cysteine ​​residue(s) of the constant domains have a free thiol group. F(ab')2 antibody fragments are the name used herein to refer to Fab' fragments having a hydroxyl group between them. Other chemical couplings of antibody fragments have been produced as pairs of Fab' fragments with transcysteines. It is also known as

[0013] The "light chains" of antibodies (immunoglobulins) from vertebrate species have the amino acid sequence of the constant domain Based on the It is possible.

[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 present invention, the Fv polypeptide is H Domains and V L A polypeptide linker is inserted between the domains. This enables the sFv to form the desired structure for antigen binding. For a review of Fv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). Please refer to.

[0015] The term "diabody" refers to a small antibody fragment that has two antigen-binding sites. The fragment is a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain. The VH-VL domains are too short to allow pairing between the two domains on the same chain. By using a linker that is long enough, the domains can pair with complementary domains on another strand. Diabodies are constrained to create two antigen-binding sites. O 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (19 93) is described in more detail.

[0016] The construction of various non-traditional antibodies is shown in FIG.

[0017] As used herein, the term "affinity" refers to the reversible binding of two agents. The equilibrium constant is expressed as the dissociation constant (Kd). The affinity is measured against unrelated amino acid sequences. At least 1-fold greater, at least 2-fold greater, or at least 3-fold greater than the affinity of the antibody Larger, at least 4 times larger, at least 5 times larger, at least 6 times larger, at least At least 7 times larger, at least 8 times larger, at least 9 times larger, at least 10 times larger, At least 20 times larger, At least 30 times larger, At least 40 times larger, At least 50 times larger Large, at least 60 times larger, at least 70 times larger, at least 80 times larger, at least 90 times larger, at least 100 times larger, or at least 1000 times larger, or The affinity of the antibody for the target protein can be, for example, about 100 nanomolar or more. (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar As used herein, the term "avi" refers to a group of animals that are a species of a single molar (fM) or larger. "Immunoimmunity" refers to the resistance of a complex of two or more agents to dissociation after dilution. "Reactive" and "preferentially binds" refer to antibodies and / or antigen-binding fragments. and are used interchangeably herein.

[0018] The term "bond" includes, for example, covalent, electrostatic, hydrophobic, and ionic bonds and / or between two molecules by hydrogen-bonding or hydrogen-bonding interactions (including interactions such as salt bridges and water bridges) MDR1-specific antibodies specifically bind to epitopes within the MDR1 polypeptide. In some embodiments, the anti-MDR1 antibody does not bind to cells that do not express MDR1. An epitope is a sequence of amino acids that binds to a target molecule. Linear epitopes formed by a sequence of amino acids or non-contiguous epitopes Non-specific binding may be a non-linear or conformational epitope. -7 Less than M binding with an affinity of, e.g., 10 -6 M, 10 -5 M, 10 -4 This refers to binding with an affinity such as M.

[0019] As used herein, the term "CDR" or "complementarity determining region" refers to a region of a heavy chain polypeptide. The term "antigen binding site" refers to the discontinuous antigen binding sites found within the variable regions of both the high and light chain polypeptides. The CDRs are hypervariable regions, also called "framework regions (FR)." The CDRs are interspersed with more conserved regions. 09-6616 (1977); Kabat et al., US Dept. of Health and Human Services, “Sequenc es of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Bi ol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996 ), where the definitions, when compared with each other, are Nevertheless, antibodies or grafted antibodies or The application of either definition to refer to a CDR of a variant thereof is not intended to be limiting as defined and understood herein. The terms used herein are intended to be within the scope of the terms defined by each of the above cited references. The amino acid residues which encompass the CDRs are set forth below in Table 1 as a comparison.

[0020] [Table 1]

[0021] As used herein, the term "framework" is used in reference to antibody variable regions. When used herein, it refers to all amino acid residues in the variable region of an antibody outside the CDR region. It is contemplated that the variable region framework is generally a non-contiguous amino acid sequence approximately 100-120 amino acids in length. Although the amino acid sequence is intended to refer only to amino acids outside the CDRs, As used herein, the term "framework region" refers to the framework regions separated by the CDRs. The VH chain is intended to mean each domain of the VH chain in the following order from N-terminus to C-terminus: It may comprise three CDRs and four FRs arranged as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, Similarly, the VL chain has three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: The FRs may include: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0022] As used herein, the term antibody refers to a tetramer of two heavy chains and two light chains. wherein the heavy and light chains are interconnected, for example, by disulfide bonds. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. The light chain constant region is composed of The variable regions of the heavy and light chains interact with antigens. The constant region of an antibody typically binds to host tissues and factors (such as the immune system). They mediate the binding of antibodies to various cells and the first component of the complement system. The term includes immunoglobulins of IgA, IgG, IgE, IgD, IgM and their subtypes. In certain embodiments, the antibody of interest is an IgG isotype, e.g., IgG1.

[0023] 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 thereby. Human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), and gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes; and Many immunoglobulin variable region genes are included. Full-length immunoglobulin light chains (approximately 25 kDa) The N-terminus (approximately 110 amino acids) contains the variable region gene and the C-terminus (approximately 214 amino acids). The full-length immunoglobulin heavy chain (approximately 50 The variable region gene (approximately 116 amino acids) is located at the N-terminus (approximately 116 amino acids) and the C-terminus (approximately 446 amino acids) is located at the At the end, one of the other constant region genes, such as gamma (encoding approximately 330 amino acids) In certain embodiments, the subject antibodies comprise a full-length immunoglobulin heavy chain and and full-length immunoglobulin light chains.

[0024] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that specifically bind to an antigen. Examples of binding fragments include: (i) Fab fragments (ii) a fragment (e.g., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); F(ab')2 fragment (two Fab fragments linked by a disulfide bridge in the hinge region) (iii) a bivalent fragment containing a Fd fragment (a VH and CH1 domain); (iv) Fv fragments (e.g., antibody fragments) (v) comprising, e.g., consisting of, a VH and a VL domain of one arm; (vi) isolated fragments (including, for example, consisting of, a VH domain); (vii) single-chain Fv (scFv) (where the VH and VL domains of a single arm of an antibody are engineered together); The VH and VL domains are paired to form monovalent molecules, linked by a synthetic linker using (viii) diabodies (including, for example, consisting of, The VH and VL domains are linked so that they are not paired to form a monovalent molecule, those comprising two scFvs, e.g. consisting of two such scFvs; pairs with the VL domain of another scFv to form a bivalent molecule).

[0025] The term "chimeric" antibody refers to an antibody in which some portions of the heavy and / or light chains are derived from a particular source or species. and the remainder of the heavy and / or light chains are derived from a different source or species. This refers to

[0026] A "human antibody" is an antibody produced by a human or human cells, or a human antibody receptor. corresponding to antibodies derived from non-human sources that utilize human antibody coding sequences, such as ribosomal or other human antibody coding sequences. This definition of a human antibody refers to a human antibody that contains non-human antigen-binding residues. Specifically excludes immunized antibodies.

[0027] The "human consensus framework" refers to the human immunoglobulin variable light (VL) or variable Represents the most commonly occurring amino acid residues in a selection of variable heavy (VH) framework sequences Framework (FR). Generally, the selection of a VL or VH sequence for a human immunoglobulin is based on: This arises from a subgroup of variable domain sequences. Generally, this subgroup of sequences is et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Pu Subgroups such as those in the publication 91-3242, Bethesda, Md. (1991), vols. 1-3 In one embodiment, for the VL, this subgroup is the In one embodiment, for VH, this subgroup is This is subgroup III in Kabat et al.

[0028] A "humanized" antibody is one that contains amino acid residues derived from non-human CDRs and human framework (FR) residues. At least a portion of the constant region of a humanized antibody is a chimeric antibody containing amino acid residues derived from For example, derived from a human antibody, such as human IgG1. The antibody molecules described herein may have variable heavy chain regions and sequences corresponding to the sequences listed in UniProt: P01857-1 Version 1. and a heavy chain comprising a human IgG1 constant region having the amino acid sequence provided. In the present disclosure, the antibody molecules comprise a variable light chain region as provided herein and a human light chain. In a preferred embodiment, the human light chain constant region is a light chain comprising a UniP rotKB / Swiss-Prot: P01834.2. In certain embodiments, the human IgG1 heavy chain constant region present in the subject antibody provides, e.g., Fc function. For example, mutations such as substitutions to modulate LALAPG effector function may be included. The mutations (L234A, L235A, and P329G) or the N297A mutation were introduced to perform antibody-dependent cellular cytotoxicity (ADAC). The substitution numbering is based on the EU numbering system. The "EU numbering system" or "EU index" generally refers to the immunoglobulin heavy chain constant Used to refer to residues within a region (e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) reported the EU Index. "U index" refers to the residue numbering of the human IgG1 EU antibody.

[0029] A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

[0030] The term "epitope" refers to a molecule that is recognized by the immune system (e.g., an antibody, a B cell, or a T cell). For example, an epitope is a specific region of an antigen to which an antibody binds. The term specifically refers to linear (continuous) and nonlinear (conformational or discontinuous) epitopes. It encompasses epitopes.

[0031] An "isolated" antibody is one that has been identified and separated and / or purified from a component of its natural environment. Contaminant components of its natural environment may affect the diagnostic or therapeutic value of the antibody. are substances that interfere with therapeutic use, such as enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, the antibody may be: (1) purified by the Lowry method; (2) greater than 90%, greater than 95%, or greater than 98% by weight of antibody, e.g., greater than 99% by weight, as measured by The use of a spinning cup sequenator allows for the identification of small N-terminal or internal amino acid sequences. (3) enough to obtain at least 15 residues, or (4) using Coomassie blue or silver staining. Sodium dodecyl sulfate-polyacrylamide gels under reducing or non-reducing conditions using The isolated antibody is purified to homogeneity by electrophoresis (SDS-PAGE). The antibody in situ within the recombinant cell is not present because at least one component of its natural environment is absent. In some instances, an isolated antibody is prepared by at least one purification step. do.

[0032] The terms "MDR1" and "ABCB1" are used interchangeably herein and refer to human MDR1 and and naturally occurring variants thereof (e.g., allelic variants). Refers to multidrug resistance cellular efflux pump proteins.

[0033] As used herein, the term "cytotoxic agent" refers to an agent that inhibits or prevents the function of cells and "Chemotherapeutic agent" refers to a substance that causes cell death or destruction and / or cell death. Cytotoxic agents, also known as "cancer agents," used to treat cancer or other diseases or disorders It may be an agent.

[0034] As used herein, the terms "treatment," "treating," and the like refer to the desired pharmacological and It means to obtain a therapeutic and / or physiological effect, which effect may be to complete the disease or its symptoms. It may be prophylactic in the sense of wholly or partially preventing and / or reducing the risk of disease and / or or may be therapeutic in the sense of partially or completely curing adverse effects resulting therefrom. As used herein, "treatment" refers to any treatment of a disease in a mammal, including a human. (a) A diagnosis of a patient who may have a predisposition to a disease but is not yet diagnosed with the disease. (b) preventing disease from occurring in an untreated subject; (c) inhibiting disease, (c) to alleviate the disease, i.e., to cause regression of the disease. To rub.

[0035] The terms "individual," "subject," "host," and "patient," used interchangeably herein, Examples include, but are not limited to, murine (rat, mouse), non-human primate, human, canine, and nematode species. This refers to mammals, including ungulates (e.g., horses, cattle, sheep, pigs, and goats).

[0036] A "therapeutically effective amount" or "effective amount" refers to an amount administered to a mammal or other subject to treat a disease. and (b) a compound that has sufficient target specificity to provide such treatment for the disease when administered to a mammal. A "therapeutically effective amount" refers to the amount of antibody that is effective in treating the disease and its severity, and the therapeutic effect of the antibody. It varies depending on the subject's age, weight, etc.

[0037] As used herein, the term "refractory" refers to a disease or condition that does not respond to treatment. As used herein, with respect to cancer, "treatment-resistant cancer" refers to a cancer or condition that does not respond to treatment. Treatment-resistant cancers may be resistant at the start of treatment or may be non-responsive to treatment. Cancers that are resistant to treatment may also be called resistant cancers.

[0038] "Biological sample" encompasses a variety of sample types obtained from an individual, including those used in diagnostic assays or can be used in monitoring assays. The definition includes blood and other biological sources. liquid samples of tissue, solid tissue samples such as biopsies or tissue cultures, or samples derived therefrom. The definition also includes cells that have been treated with a reagent, solubilized, or cloned, and their progeny. or after its procurement, such as enrichment for a particular component, e.g., polynucleotides. The term "biological sample" encompasses clinical samples, as well as samples that have been manipulated in any way. Also encompassed are cells in culture, cell supernatants, cell lysates, serum, plasma, body fluid, and tissue samples.

[0039] The percent identity between a pair of sequences is calculated by multiplying the number of matches in the pair by 100 and This can be calculated by dividing the length of the region being addressed (including gaps). Identity scoring counts only exact matches and does not consider the degree of amino acid similarity. The length includes only internal gaps, not gaps at the end of the sequence. Percent identity = (matches x 100) / length of aligned region (including gaps) Mel)

[0040] The phrase "conservative amino acid substitution" refers to a substitution of an amino acid residue within the following group: :1) L, I, M, V, F;2) R, K;3) F, Y, H, W, R;4) G, A, T, S;5) Q, N;6) D, E. Conservative amino acid substitutions replace one or more amino acids in a protein with similar acidic, basic, or By substituting amino acids with side chains that have charge, polarity, or size, The activity of the protein can be preserved.

[0041] The term "vector" refers to any vector used to transfer protein coding information to a host cell. any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) means.

[0042] The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell. and capable of inducing (in conjunction with a host cell) expression of one or more operably linked heterologous coding regions. An expression construct refers to a vector containing nucleic acid sequences that induce and / or regulate the expression of an operably expressed gene. Transcription, translation, and, if introns are present, RNA splicing of functionally linked coding regions. These sequences may include, but are not limited to, sequences that affect or regulate lysing. stomach.

[0043] The term "stimulation" refers to the interaction between a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) and its cognate ligand (e.g., a The primary response is induced by binding to a target antigen (or tumor antigen in the case of CARs), which For example, signaling through the TCR / CD3 complex or signaling of the appropriate NK receptor or CAR. signaling events, including but not limited to signaling through the nucleotransduction domain Stimuli can mediate changes in the expression of specific molecules.

[0044] The term "stimulatory molecule" refers to a molecule that stimulates at least one aspect of an immune cell signaling pathway. Immune cell signaling sequences that regulate immune cell activation in a toxic manner. (e.g., T cells, NK cells, B cells). The signal is initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. It is the primary signal that mediates T cell responses (including but not limited to proliferation, activation, and differentiation). Primary cytoplasmic signaling sequences ("primary") acting in a stimulatory manner. The immunoreceptor tyrosine-based activation motif (Immu) is a tyrosine-based signaling domain (also called the "secondary signaling domain"). signal transduction pathway known as ITAMs (Inhibitor Tyrosine-Based Activation Motifs) The IT containing a cytoplasmic signaling sequence may be particularly useful in the present invention. Examples of AMs include CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and and DAP12.

[0045] The term "costimulatory molecule" refers to a molecule that specifically binds to a costimulatory ligand, thereby stimulating T cell proliferation. Cognate binding on T cells mediates costimulatory responses (such as, but not limited to, proliferation) Costimulatory molecules are molecules that bind to antigen receptors or their receptors, which contribute to an efficient immune response. Costimulatory molecules are cell surface molecules other than ligands. Costimulatory molecules include, but are not limited to, MHC class II receptors. RasI molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, These include LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0046] The term "autologous" refers to a material derived from the same individual to which it is later reintroduced. It refers to a substance that

[0047] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain controls the immune effector functions of CAR-containing cells (e.g., CAR-T cells). - generate signals that promote immune effector function, e.g., in CAR-T cells Examples of these include cytolytic and helper activities, which increase cytokine secretion. include.

[0048] As used herein, the term "immune effector cell" refers to, e.g., an immune effector - Refers to cells that participate in the immune response in promoting the response. Examples of immune effector cells include , T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer cells, NK cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes Included.

[0049] Guidance on substitutions, insertions or deletions is provided for amino acid sequences of proteins from different species. Based on sequence alignment or identification of multiple proteins with the same or similar functions The sequence may be derived from a consensus sequence based on the protein.

[0050] Detailed Description Antibodies that target the cellular efflux pump MDR1 are provided. Also provided are pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits encoding the compounds. Detecting the presence or absence of MDR1 expression in cells (e.g., tumor cells) and the level of MDR1 expression. Also disclosed are methods of using the antibodies to inhibit MDR1 function and / or inhibit MDR1 function. Additionally, multispecific antibodies that bind to MDR1 and TAAs are provided. A method of treating a tumor comprising administering to a subject an anti-MDR1 antibody disclosed herein, or a combination of MDR1 and a tumor-associated antigen, Also provided are methods comprising administering to a subject a multispecific antibody that targets both

[0051] Before describing the present invention in more detail, it is to be understood that the invention is not limited to the particular embodiments described. It should be understood that such embodiments may, of course, vary. The scope of the invention is limited only by the appended claims, which are used herein. The terminology used is for the purpose of describing particular embodiments only and is not intended to be limiting. Please understand that this is not a

[0052] When a range of numerical values ​​is provided, each intervening value is included within the range unless the context clearly dictates otherwise. Between the upper and lower limits of and any other stated or intervening value within that stated range, It is understood that all tenths of a unit are encompassed within the present invention. The upper and lower limits of a range may independently be included in the smaller ranges and may be used interchangeably within the stated range. Any specifically excluded limitation within the scope of the present invention is encompassed within the scope of the present invention. Where the stated range includes one or both of the limits, The range not included is also included in the present invention.

[0053] Certain ranges are presented by numerical values ​​preceded by the term "about." is used herein to refer to the exact number that follows the term, as well as the number that follows the term. Used to provide a literal justification for a number being close or an approximation of that number. In determining whether a number is near or approximate to a specifically stated number, close or approximate A number that is approximate but not specifically stated is one that is within the context in which it is presented. , may be substantially equivalent to the numbers specifically stated.

[0054] Unless otherwise defined, all technical and scientific terms used herein are , have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Any methods and materials similar or equivalent to those described herein can also be used to practice the present invention. Representative exemplary methods and materials are described here. Explain the fees.

[0055] All publications and patents cited herein are to be construed as though each individual publication or patent were incorporated by reference. and all other elements of the present invention are incorporated by reference as if specifically and individually indicated to be incorporated by reference. The methods and / or materials related to which the publication is incorporated herein and to which it is cited are The materials disclosed and described herein are incorporated by reference. for its disclosure prior to the filing date and that the present invention is not entitled to antedate such publication. Further, any publication dates provided may not be used to identify, represent, or imply any endorsement by, any person or entity. The dates of publication may be different from the actual publication dates which should be verified by individual investors.

[0056] As used in this specification and the appended claims, the singular forms "a," "an," "an" and "an" are used interchangeably. " and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, claims should be drafted to exclude optional elements. Therefore, this statement should be read in conjunction with the recitation of claim elements. Use of exclusive terms such as "solely" or "only" or use of the limitation "except" It is intended to serve as a basis for

[0057] As will be apparent to those skilled in the art upon reading this disclosure, the particular implementations described and illustrated herein may be Each of the aspects may be combined with other embodiments without departing from the scope or spirit of the invention. The present invention provides a method for the production of a computer-readable recording medium, which is a separate component and feature that can be easily separated or combined from any of the features of the present invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible. The steps may be performed in any order.

[0058] The methods and compositions are described with functional descriptions for grammatical flow or As will be described, the claims are expressly formulated under 35 U.S.C. § 112(f). Unless otherwise specified, the invention is not necessarily limited by the construction of a "means" or "step" limitation. The meaning and equivalence of the definitions provided by the claims under the doctrine of equivalents should not be interpreted as meaning or equivalence of the The full scope of the invention should be given, and the claims should be If expressly formulated in the It should be clearly understood that the

[0059] antibody As summarized above, the present disclosure relates to P-glycoprotein 1 (Pgp), also known as ABCB1. The present invention provides an antibody that binds to the cellular efflux pump MDR1.

[0060] MDR1, also known as P-glycoprotein 1 (Pgp) or ABCB1, is an ATP-binding cassette subunit The ABCB1 gene is expressed by the family B member 1 (ABCB1) gene, which mediates drug accumulation in multidrug-resistant cells. Anti-MDR1 antibodies are used herein to refer to anti-Pg p, also called anti-ABCB1 or anti-B1 antibody.

[0061] In some embodiments, the antibodies disclosed herein target a nucleotide sequence on the extracellular domain of MDR1. In some embodiments, the anti-MDR1 antibodies of the present disclosure bind to an epitope comprising one or more sites. In some embodiments, the anti-MDR1 antibodies of the present disclosure bind to human MDR1 on the cell surface of human cells. In some embodiments, the antibodies disclosed herein bind to human MDR1 expressed in human MDR1. and binding to an epitope comprising one or more sites on the extracellular domain (ECD) of MDR1, wherein the EC D is the following sequence: MMLVFGEMTDIFANAGNLEDLMSNITNRSDINDTGFFMNLEEDMTRYAY (SEQ ID NO: 1) which sequence may comprise the amino acid sequence of the human MDR1 sequence having accession number NP_000918. corresponding to residues 68 to 116 of human MDR1. Binds to an epitope comprising one or more sites on the extracellular domain (ECD), where the ECD is a sequence The sequence TRGWKL (SEQ ID NO: 2) can be found in the human genome having the accession number NP_000918. In one embodiment, the present invention relates to a method for the treatment of MDR1 comprising administering to a subject a therapeutic agent ... The antibody binds to epitopes comprising one or more sites on the extracellular domain (ECD) of human MDR1. wherein the ECD may comprise the sequence GTTLVLSGEYSIGQ (SEQ ID NO: 3), which sequence is It corresponds to amino acid residues 317 to 330 of the human MDR1 sequence having the sequence number NP_000918. In such a manner, the antibodies disclosed herein bind to one or more of the extracellular domains (ECDs) of human MDR1. The ECD binds to an epitope containing the sequence SKIIGVFTRIDDPETKRQNSNLFS (sequence 4), which is a sequence of the human MDR1 sequence having accession number NP_000918. corresponding to amino acid residues 733 to 756. In certain embodiments, the antibodies disclosed herein comprise: binds to an epitope comprising one or more sites on the extracellular domain (ECD) of human MDR1, The ECD can comprise the sequence RFGAYLVAHKLMSFED (SEQ ID NO: 5), which is located in Accession No. This corresponds to amino acid residues 958 to 973 of the human MDR1 sequence with P_000918.

[0062] The anti-MDR1 antibodies of the present disclosure may have one or more of the following properties. i) inhibiting efflux from MDR1; ii) increasing the sensitivity of cancer cells to treatment with chemotherapeutic agents, thereby Reduce the IC50 of the therapeutic agent by at least 5, 10, 50, or 100-fold; iii) binds to human and cynomolgus monkey MDR1; iv) does not cross-react with CD47; v) effective in in vitro cell killing assays; vi) be effective in inhibiting tumor growth even in the absence of chemotherapy; vii) preferentially binds to MDR1 mutants constrained in an open conformation; viii) preferentially binds to MDR1 mutants constrained in a closed conformation; and ix) Preferentially bind to regions outside extracellular loop 1 of human MDR1 or bind to the cellular domain of human MDR1 Binds to extracellular loop 1 and extracellular loop 4.

[0063] In certain embodiments, in addition to having one or more of the above characteristics i)-ix), The antibody has an EC50 of 100 nM or less, for example, 100 nM to 4 nM, 80 nM to 4 nM, 60 nM to 4 nM, 40 nM The saturation concentration can be 0.01 to 0.04 nM, ... In certain embodiments, in addition to having one or more of the above characteristics i) to ix), the present invention The antibodies shown have EC50s at least as low as those of anti-MDR1 antibodies such as 15D3, MRK16, MM4, and / or UIC2. may have an EC50 that is half as high.

[0064] The EC50 of a test antibody can be measured by flow cytometry or ELISA. Flow cytometry was performed by measuring MDR1 (e.g., human wild-type) in flow cytometry buffer. contacting a cell expressing MDR1 or a mutant MDR1 with an antibody (wherein the antibody The antibody is then allowed to stand for a sufficient time (e.g., 10 minutes to 1 hour) for the antibody to bind to the cells. This may include incubating at room temperature or 4°C for 10 minutes (20 minutes). Optionally, the cells may be washed to remove non-specifically bound antibodies and / or The cells may be contacted with a fluorescently labeled secondary antibody that specifically binds to the test antibody. After the injection, the fluorescently labeled secondary antibody may be removed and the cells washed. The cells can be sorted by cytometry and the number of cells bound to the fluorescently labeled secondary antibody can be counted. The concentration that provides half the maximal response (e.g., half the maximal fluorescence intensity) is determined as the EC50. In a variation of the flow cytometry assay, cells expressing MDR1 The cells may be 293T cells expressing the IL-1 agonist.

[0065] The IC50 of a test antibody can be determined by measuring the inhibition of cell proliferation. 50 is determined by using the test antibody alone to determine the concentration of antibody that produces a half-maximal response. The IC50 of a chemotherapeutic agent can be measured by IC50, which determines the effect of the antibody on the chemotherapeutic agent. The chemotherapeutic agent is vincristine, and the antibody can be measured in the absence and presence of the test antibody. The cells may be a cancer cell line. The cancer cell line may be a human acute lymphoblastic leukemia ( N6 / ADR, a doxorubicin-selected B1-positive variant of the ALL cell line NALM6. DR cells are also called NALM6 / ADR cells. The cells are used in the determination of IC50 of an antibody. The cells may be contacted with an antibody and a chemotherapeutic agent, where the antibody is tested in serial dilutions. The effect of the antibody on the IC50 of the drug can be determined, where the drug is continuously The cells are incubated at 37°C for a period of time (e.g., 24 hr to 84 hr), and the standard Cell viability can be assessed using standard reagents and methods. Increase the sensitivity of cancer cells to treatment with chemotherapeutic agents, thereby The IC50 may be reduced by at least 5-fold. The cancer cells may be N6 / ADR. The chemotherapeutic agent may be vincristine. In some embodiments, the antibodies of the present disclosure inhibit the growth of N6 / ADR cancer cells. The IC50 of vincristine for times or more, 9 times or more, or 10 times or more, for example, 5 times to 50 times or more, for example, at least 60, 70, 80 , 90, or 100-fold reduction. B1.80, KB1.261 and KB1.263 inhibit vincristine to inhibit the proliferation of N6 / ADR cancer cells at least 400-fold reduction in the IC50 of See Figures 4A to 4D and Table 7.

[0066] In certain embodiments, the anti-MDR1 antibody binds to both human and cynomolgus MDR-1. This property can be exploited in determining the safety of antibodies in animal models. can.

[0067] In certain embodiments, the anti-MDR1 antibodies disclosed herein are specific for MDR1 and are not specific for other For example, the antibodies disclosed herein do not exhibit significant binding to the antigen. In contrast, it does not cross-react with the tumor-associated antigen CD47.

[0068] In certain embodiments, the anti-MDR1 in vitro cell killing activity of the antibodies disclosed herein is , may be superior to those observed for MRK16, MM4, and / or UIC2. For example, the antibodies disclosed herein may have a binding activity that is at least twice as strong as that of 15D3, MRK16, MM4, and / or UIC2. It may have in vitro cell killing activity.

[0069] In certain embodiments, the antibodies provided herein comprise MDR1 constrained in an open conformation. The MDR1 mutants are human or mouse MDR1 mutants containing the substitutions E556Q and E1201Q. The numbering of amino acid positions may be based on that of human MDR1. Such antibodies are less abundant compared to wild-type MDR1 or MDR1 mutants constrained in a closed conformation. with at least 2x (e.g., 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, or more) affinity , can bind to MDR1 mutants constrained in the open conformation.

[0070] In certain embodiments, the antibodies provided herein comprise MDR1 constrained in a closed conformation. The MDR1 mutants were human or cynomolgus monkey MDR1 containing the following substitutions: R1 can be: (i) K433M, S434A, K1076M, S1077A; (ii) K433M, S434A, Q475A, K1076M , S1077A, Q1118A; and / or (iii) K433M, S434A, Q475A, R588E, K1076M, S1077 A, Q1118A, R1233E. Here, the numbering of amino acid positions is based on human MDR1. Such antibodies inhibit at least 100% of the MDR1 signaling pathway compared to wild-type MDR1 or MDR1 mutants constrained in the open conformation. with twice (e.g., three, four, five, six, seven, eight, nine, ten, or more) times greater affinity , can bind to MDR1 mutants constrained in a closed conformation.

[0071] In certain embodiments, the antibodies provided herein comprise a deletion in loop 1 in the ECD. The deletions consist of deletions of amino acid residues 82-99 or 79-102. wherein the numbering of amino acid positions is based on human MDR1. at least as compared to wild-type MDR1 or MDR1 mutants constrained in the open or closed conformation with twice (e.g., three, four, five, six, seven, eight, nine, ten, or more) times greater affinity , can bind to MDR1 mutants containing deletions in loop 1.

[0072] In certain embodiments, the antibodies provided herein are directed to extracellular loop 1 of human MDR1 and Binds to an epitope comprising extracellular loop 4. Examples of such antibodies include those listed in Table 2. Anti-MDR1 antibodies B1.228B, B1.198, B1.223, B1.219, B1.184, B1.207, B1.201, B1.129, B Examples of antibodies include antibodies having a VH chain containing HCDRs 1 to 3 of B1.273 or B1.236 and a VL chain containing LCDRs 1 to 3 of B1.273 or B1.236. It can be obtained.

[0073] In certain embodiments, the antibodies provided herein are monospecific bivalent anti-MDR1 antibodies In some embodiments, the monospecific bivalent anti-MDR1 antibodies of the present disclosure include the anti-MDR1 antibodies 15D3, MRK16 At least one or two of HCDRs 1 to 3 and LCDRs 1 to 3 present in MM4 and UIC2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12.

[0074] In certain embodiments, the antibodies of the disclosure comprise the variable heavy (VH) regions and The heavy chain complementarity-determining regions (HCDRs) and light chain CDRs (LCDs) of the pair of variable and light chain (VL) regions are R) for binding to MDR1. For example, in one embodiment, The anti-MDR1 antibodies compete with the B1.201 antibody listed in Table 2 for binding to MDR1.

[0075] In certain embodiments, the antibody comprises HCDR1, HCDR2, and HCDR3 of the VH region of an antibody listed in Table 2. In certain embodiments, HCDR1, HCDR2, and HCDR3 are defined according to the Kabat nomenclature. For example, in one embodiment, the B1.27 antibody, which is described in Table 2 for binding to MDR1, is The anti-MDR1 antibodies of the present disclosure that compete with the antibody bind to HCDR1, HCDR2, and HCDR3 of the VH region of the B1.27 antibody. include.

[0076] Optional assay to determine whether a first antibody competes with a second antibody for binding to MDR1. Any suitable approach can be used. Whether a compound "competes" with an antibody can be readily 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 the first antibody can be bound to a solid support. A sample of a second antibody is added, which may be capable of competing with the antibody. The labeled and unlabeled antibodies bind to different sites on the compound. When the antibody is labeled, it reacts with the antibody regardless of whether or not a suspected competitor is present. However, if the interaction sites are identical or overlapping, If the labeled antibody is present, the unlabeled antibody will compete and reduce the amount of labeled antibody that binds to the antigen. If there is an excess of unlabeled antibody, very little, if any, of the labeled antibody will bind. .

[0077] For purposes of this disclosure, a competing antibody is one that inhibits the binding of the antibody to the compound by about 30% or more, about 40% or more, , about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or a reduction of about 99% or more. Details of this are well known in the art and are described, for example, in Harlow and Lane, Antibodies ies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor r, New York, 1988, pp. 567-569, 1988, ISBN 0-87969-314-2. Such assays can be performed quantitatively using purified antibodies. by titrating the antibody against itself (i.e., the same antibody is both labeled and competed) A standard curve (used for both the assay and the target) can be established. The ability of unlabeled competing antibodies to inhibit binding can be titrated. The results are plotted. and compare the concentrations required to achieve the desired degree of binding inhibition.

[0078] In certain embodiments, the antibody molecule that binds to multidrug resistance protein 1 (MDR1) is (i) an antibody molecule selected from the group consisting of: HCDRs 1-3 and 2 of the variable heavy (VH) and variable light (VL) region pairs of the described antibodies (ii) HCDRs 1 to 3 of the VH region of an antibody listed in Table 2; (iii) HCDRs 1 to 3 of the VH region of an antibody listed in Table 2; The VH region of the described antibody includes LCDRs 1 to 3. HCDRs and LCDRs are based on the Kabat nomenclature. It can be defined.

[0079] The term "antibody molecule" includes antibodies as defined herein and their antigen-binding In some embodiments, an antibody molecule comprises two variable light (VL) chains and two variable heavy (VH) chains. In some embodiments, the antibody molecule also comprises heavy and light chain constant regions. The heavy and light chain constant regions can be derived from a human antibody, such as a human IgG1 antibody. The (HC) constant region is modified to contain mutations that reduce antibody-dependent cellular cytotoxicity (ADCC). Additionally or alternatively, the two VH chains may each be a different human IgG1 The individual human IgG1 HC constant regions are linked to the HC constant regions, and the individual human IgG1 HC constant regions are linked to the different human IgG1 HC constant regions. Such an HC region may have substitutions that favor dimer formation. In certain embodiments where the antibody molecule is a bispecific antibody molecule, In this regard, one of the human IgG1 HC constant regions contains one or more amino acids with positively charged side chains. The other human IgG1 HC constant region contains substitutions that introduce one or more negatively charged side chains. It contains substitutions that introduce amino acids that may promote the formation of dimers between the two different HCs.

[0080] In certain embodiments, the antibody molecule comprises a pair of VH and VL regions of an antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-27 antibody described above. The antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-28 antibody described in Table 2. In this embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs of the B1-30 antibody described in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 of the B1-39 antibody described in Table 2. and / or LCDRs 1-3. In one embodiment, the antibody molecule comprises any of B1-, B2-, and B3-LCDRs listed in Table 2. HCDRs 1-3 and / or LCDRs 1-3 of antibody 85. In one embodiment, the antibody molecule comprises: In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-99 antibody as set forth in Table 2. The antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-178 antibody described in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and 2 of the B1-179 antibody as set forth in Table 2. In one embodiment, the antibody molecule comprises the B1-184 antibody described in Table 2 and / or LCDRs 1-3. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the same amino acid sequence as in Table 2. In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-188 antibody described herein. The antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-193b antibody described in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or HCDRs 1-3 of the B1-197 antibody described in Table 2. or LCDRs 1-3. In one embodiment, the antibody molecule comprises the H of the B1-198 antibody described in Table 2. In one embodiment, the antibody molecule comprises CDRs 1-3 and / or LCDRs 1-3. In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-201 antibody. The antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-207 antibody described in Table 2. In this embodiment, the antibody molecule comprises HCDRs 1-3 and / or LC of the B1-223 antibody as described in Table 2. Includes DR 1-3.

[0081] In certain embodiments, the antibody comprises the HCDRs of the VH and VL regions of the antibody pairs set forth in Table 2. 1 to 3 and / or LCDR 1 to 3, and the IC50 of a chemotherapeutic agent is increased by 5 times or more, 6 times or more, or 7 times or more; 8 times or more, 9 times or more, or 10 times or more, for example, 5 times to 50 times or more, for example, at least The anti-MDR1 antibodies B1-207, B1-223, and K B1-225, KB1.116, KB1.80, KB1.261 and KB 1.263 inhibited the proliferation of N6 / ADR cancer cells. The IC50 of vincristine for α-glucanase was increased by at least 400-fold, from 4118 pM to 10.3 pM or more. See Figures 4A to 4D and Table 7.

[0082] EC50 is the antibody concentration that produces 50% of the maximum response (e.g., the response is the binding of the antibody to the antigen). In one embodiment, the antibody may have an EC50 of 100 nM or less, and / or an anti-MDR1 antibody. EC50 of at least half of the EC50 of the corresponding agonist (e.g., 15D3, MRK16, MM4, and / or UIC2) 50 and comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-27 antibody as set forth in Table 2. In such cases, the antibody has an EC50 of 100 nM or less, and / or a specific activity similar to that of an anti-MDR1 antibody (e.g., 15D3, MRK16 , MM4, and / or UIC2) and are listed in Table 2. In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the modified B1-28 antibody. nM or lower EC50, and / or anti-MDR1 antibodies (e.g., 15D3, MRK16, MM4, and / or has an EC50 that is at least half the EC50 of the B1-30 antibody (e.g., UIC2), and In one embodiment, the antibody has an EC50 of 100 nM or less, and and / or the EC50 of anti-MDR1 antibodies (e.g., 15D3, MRK16, MM4, and / or UIC2) having an EC50 that is at least half that of HCDRs 1-3 and / or HCDRs 1-3 of the B1-39 antibody listed in Table 2 comprises LCDRs 1-3. In one embodiment, the antibody has an EC50 of 100 nM or less and / or an anti-MDR1 At least half the EC50 of the antibody (e.g., 15D3, MRK16, MM4, and / or UIC2). and contains HCDRs 1 to 3 and / or LCDRs 1 to 3 of the B1-85 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less, and / or a binding affinity to an anti-MDR1 antibody (e.g., 15D3, MR have an EC50 that is at least half of the EC50 of the agonist (e.g., K16, MM4, and / or UIC2), and In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-99 antibody described. , an EC50 of 100 nM or less, and / or an anti-MDR1 antibody (e.g., 15D3, MRK16, MM4, and / or or UIC2), and In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the same antibody. In one embodiment, the antibody has an EC50 of 100 nM or less. and / or anti-MDR1 antibodies (e.g., 15D3, MRK16, MM4, and / or UIC2). and having an EC50 that is at least half of the EC50 of HCDRs 1-3 and HCDRs 1-3 of the B1-179 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less, and / or LCDRs 1-3. At least half the EC50 of the anti-MDR1 antibody (e.g., 15D3, MRK16, MM4, and / or UIC2) HCDRs 1-3 and / or LCDRs 1-3 of the B1-184 antibody as described in Table 2, having an EC50 of 1 min. In one embodiment, the antibody has an EC50 of 100 nM or less, and / or a similar activity to an anti-MDR1 antibody (e.g., have an EC50 that is at least half of the EC50 of In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-188 antibody as set forth in Table 2. , the antibody has an EC50 of 100 nM or less, and / or an anti-MDR1 antibody (e.g., 15D3, MRK16, MM4, and / or UIC2), and In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the 1-193b antibody. and / or anti-MDR1 antibodies (e.g., 15D3, MRK16, MM4, and / or UIC2 and HCDR 1 of the B1-197 antibody listed in Table 2. In one embodiment, the antibody comprises an EC50 of 100 nM or less, and / or LCDRs 1-3. Alternatively, a lower EC50 value of an anti-MDR1 antibody (e.g., 15D3, MRK16, MM4, and / or UIC2) may be used. and having an EC50 that is at least half that of HCDRs 1 to 3 and / or LC of the B1-198 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less, and / or an anti-MDR1 antibody. EC50 of at least half of the EC50 of the corresponding agonist (e.g., 15D3, MRK16, MM4, and / or UIC2) 50 and contains HCDRs 1-3 and / or LCDRs 1-3 of the B1-201 antibody as set forth in Table 2. In embodiments, the antibody has an EC50 of 100 nM or less, and / or a cytotoxicity profile similar to that of an anti-MDR1 antibody (e.g., 15D3, MRK1 6, MM4, and / or UIC2), and have an EC50 that is at least half of the EC50 of In one embodiment, the antibody comprises HCDRs 1-3 and / or LCDRs 1-3 of the B1-207 antibody described above. EC50 of 100 nM or less, and / or anti-MDR1 antibodies (e.g., 15D3, MRK16, MM4, and / or or UIC2), and the B1-223 antibody described in Table 2. In certain embodiments, the antibodies of the disclosure comprise HCDRs 1-3 and / or LCDRs 1-3 of When measured using FACS of the 293T cell line expressing human MDR1, it has an EC50 of 4 to 10 nM. In certain embodiments, 4 to 10 nM of MDR1 is measured using FACS of a 293T cell line expressing human MDR1. Antibodies of the present disclosure having EC50 include B1-28 antibody, B1-184 antibody, B1-197 antibody, B1-28 antibody, B1-198 antibody, B1-199 antibody, B1-200 antibody, B1-201 antibody, B1-202 antibody, B1-203 antibody, B1-204 antibody, B1-205 antibody, B1-206 antibody, B1-207 antibody, B1-2 HCDRs 1 to 3 and LCD of B1-201 antibody, B1-207 antibody, B1-223 antibody, B1-85 antibody, or B1-30 antibody Including R 1-3. See, for example, Figure 3D.

[0083] In some embodiments, the antibody, upon binding to a cell expressing MDR1, inhibits cellular MDR1 protein. Therefore, the antibodies of the present disclosure can inhibit the efflux by the MDR1 protein. This can include, for example, an increase in efflux of 5% or more compared to efflux by MDR1 in the absence of the antibody. This may include cases where the value decreases (e.g., by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more). In some embodiments, the percentage of the saturation is 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Therefore, when bound to cells expressing MDR1, the antibody inhibits other mechanisms (e.g., uptake of chemotherapeutic agents). by increasing uptake and / or causing MDR1 leakage, which may reduce cell viability. This can interfere with the action of MDR1.

[0084] In certain embodiments, an antibody of the disclosure comprises a VH chain comprising HCDRs 1-3 of the B1-28 antibody, and It comprises a VL chain comprising LCDRs 1 to 3 of the antibody.

[0085] In certain embodiments, an antibody of the disclosure comprises a VH chain comprising HCDRs 1-3 of the B1-261 antibody, and and a VL chain comprising LCDRs 1 to 3 of the B1-261 antibody.

[0086] In some embodiments, the antibodies of the present disclosure bind to both human ABCB1 and cynomolgus monkey ABCB1. Such an antibody comprises a VH chain containing HCDRs 1 to 3 of the B1-28 antibody and a VH chain containing LCDR 1 of the B1-28 antibody. a VL chain comprising HCDRs 1 to 3 of the B1-261 antibody, or a VH chain comprising HCDRs 1 to 3 of the B1-261 antibody, and LCDRs 1 to 3 of the B1-261 antibody In certain embodiments, the antibody may comprise a VL chain comprising the VH chain of the B1-28 antibody and the VL chain of the B1-2 The VL chain of the B1-8 antibody, or the VH chain of the B1-261 antibody and the VL chain of the B1-261 antibody, may be included.

[0087] In certain embodiments, an antibody of the disclosure comprises a VH chain comprising HCDRs 1-3 of the B1-129 antibody, and and a VL chain comprising LCDRs 1 to 3 of the B1-129 antibody.

[0088] In certain embodiments, the antibodies of the present disclosure bind to loop 1 in the ECD of ABCB1. In this embodiment, the antibody comprises a VH chain comprising HCDRs 1-3 of the B1-129 antibody, and a L In certain embodiments, the antibody may comprise a VL chain comprising CDRs 1 to 3. In certain embodiments, the antibody is the B1-129 antibody. and the VL chain of the B1-129 antibody.

[0089] In certain embodiments, an antibody of the disclosure comprises a VH chain comprising HCDRs 1-3 of the B1-225 antibody, and 5 antibody and a VL chain comprising LCDRs 1 to 3.

[0090] In certain embodiments, an antibody of the disclosure comprises a VH chain comprising HCDRs 1-3 of the B1-223 antibody, and and a VL chain comprising LCDRs 1 to 3 of the B1-223 antibody.

[0091] In some embodiments, antibodies of the present disclosure that specifically bind to human MDR1 include those antibodies listed in Table 2. The present invention includes the HCDR1, HCDR2, and HCDR3 sequences of the corresponding amino acid sequences, and the LCDR1, LCDR2, and LCDR3 sequences of the corresponding amino acid sequences. In addition to binding to human MDR1, the antibodies provided herein bind to mouse, monkey, and chinese MDR1. It can bind to MDR1 from other mammalian species, such as pansy.

[0092] Table 2: From left to right, Column 1: Anti-MDR1 antibody name, Column 2: VH region, Column 3: HCDR1, Column 4: HCDR2 , 5th column: HCDR3, 6th column: VL area, 7th column: LCDR1, 8th column: LCDR2, 9th column: LCDR3.

[0093] [Table 2] JPEG2026041756000004.jpg247162JPEG2026041756000005.jpg247163JPEG2026041756000006.jpg246163JPEG2026041756000007.jpg248163 JPEG2026041756000008.jpg248162JPEG2026041756000009.jpg248163JPEG2026041756000010.jpg247163JPEG2026041756000011.jpg247163 JPEG2026041756000012.jpg246163JPEG2026041756000013.jpg248163JPEG2026041756000014.jpg243170JPEG2026041756000015.jpg243170 JPEG2026041756000016.jpg239169JPEG2026041756000017.jpg248170JPEG2026041756000018.jpg247165JPEG2026041756000019.jpg247144

[0094] The antibodies listed in Table 2 were generated in either mice or rats.

[0095] The anti-MDR1 antibodies listed in Table 2 are also referred to herein as anti-KPB1 or B1 antibodies and are listed in Table 2. The antibody may be referenced by its listed antibody number, which indicates that the antibody binds to KPB1 or "B1." To indicate that the product is a It may further include:

[0096] In one embodiment, the antibody comprises a VL region and a VH region present in separate polypeptides. in other embodiments, the VL region and the VH region are comprised within a single polypeptide.

[0097] The antibodies of the present disclosure may comprise a humanized light chain, a humanized heavy chain, or both.

[0098] The antibodies of the present disclosure may be any of an Ig monomer, an Fab fragment, an F(ab')2 fragment, an Fd fragment, an Ig monomer, an Ig fragment ... The antibody may be selected from the group consisting of a dAb, a scFv, a scAb, a dAb, and an Fv.

[0099] In certain embodiments, the antibody comprises a VH chain comprising the HCDRs of a VH chain listed in Table 2 and the MRK16 antibody. The MRK16 antibody, the 15D3 antibody, or the UIC2 antibody may comprise a VL chain containing the LCDR of the MRK16 antibody, the 15D3 antibody, or the UIC2 antibody. The VL chain sequences of the UIC2 antibody are as follows:

[0100] MRK16 VL chain: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO:283)

[0101] 15D3 VL chain: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:284)

[0102] UIC2 VL chain: DVVMTQTPRSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHIPPWTFGGGTKLDIK (SEQ ID NO:285)

[0103] In certain embodiments, the antibody comprises a VL chain comprising the LCDR of a VL chain listed in Table 2 and the MRK16 antibody. The VH chain may comprise the HCDRs of the MRK16 antibody, the 15D3 antibody, or the UIC2 antibody. The VH chain sequences of the UIC2 antibody are as follows:

[0104] MRK16 VH chain: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLY LQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA (SEQ ID NO:286)

[0105] 15D3 VH chain: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:287)

[0106] UIC2 VH chain: AVQLQQSGPELVKTGASVKISCKASGYSFSNYYIHWVKQSHGKSLEWIGFISCYNGATFYNQKFKGKATFTVDTSSSTAY MKFNSLTFEDSAVYYCARLPIQFGNFYPMDYWGQGTSVTVSS (SEQ ID NO:288)

[0107] In certain antibodies, the antibodies are monospecific bivalent antibodies.

[0108] In certain embodiments, the antibodies of the present disclosure target at least two different target proteins. Multispecific antibodies are capable of binding to at least two different epitopes. The number of different target proteins and therefore different epitopes bound by an antibody can vary. , two (i.e., bispecific), three (trispecific), four or more.

[0109] In certain embodiments, the antibodies of the present disclosure are capable of binding to at least two different epitopes. It is a multispecific antibody that can be used with any of the following epitopes: one epitope is on MDR1 (e.g., human MDR1) and the other is on MDR1. One epitope is a tumor-associated antigen (TAA). TAAs are often overexpressed in cancer cells. For example, a TAA may be any antigen known to be detectable in normal cells. The antigen may be an antigen that is expressed in cancer cells but not at normal levels, where the antigen is expressed in normal cells and Cancer cells are of the same cell type, e.g., epithelial cells. For example, TAAs are expressed as neoantigens. ), which is a sequence of the encoded protein compared to the amino acid sequence of the native protein. A class of tumor antigens that arise from tumor-specific mutation(s) that alter the amino acid sequence. In other embodiments, the TAA is expressed in normal cells but at higher levels in cancer cells. TAAs are antigens expressed on the cell surface of mammalian cancer cells. In certain embodiments, the TAA can be CD47, Her2, or PDL1.

[0110] In certain embodiments, the multispecific antibody is used to inhibit the sensitivity of cancer cells to treatment with a chemotherapeutic agent. thereby increasing the chemotherapeutic activity of the chemotherapeutic agent when co-administered with an anti-MDR1 monospecific bivalent antibody. the IC50 of the chemotherapeutic agent when co-administered with the multispecific antibody is at least The IC50 can be measured by the methods provided herein. The chemotherapeutic agent may be vincristine. The cancer cells are drug-resistant cancer cells, N6 / ADR. In some embodiments, the multispecific antibodies of the present disclosure inhibit the IC50 of a chemotherapeutic agent by 5-fold or more, e.g., For example, by reducing it by 5-10 times, for example, by 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more. obtain.

[0111] In certain embodiments, the multispecific antibody may have in vivo cell killing activity, e.g., vincristine. There may be a reduction in tumor volume without the administration of chemotherapeutic agents such as cristatine.

[0112] In certain embodiments, the multispecific antibody is a multidrug resistance protein 1 (MDR1) and tumor-associated The antibody molecule may be a bispecific antibody molecule that binds to an antigen (TAA), the antibody molecule comprising two identical variable light chains. a first variable heavy (VL) chain, a first variable heavy (VH) chain, and a second variable heavy (VH) chain, wherein the VL chains each have a binding domain specific to MDR1. The first VH chain contains an antigen-binding site for MDR1, and the second VH chain contains an antigen-binding site for TA The second VH chain contains an antigen-binding site for TAA when paired with one of the light chains. Combine.

[0113] In certain embodiments, the antigen-binding sites of the two VL chains are identical to the light chain CDRs 1 and 2 of the antibodies listed in Table 2. In a specific embodiment, the antigen-binding sites of the two VL chains comprise the following amino acids: Amino acid sequence: LWVPGSTGDVLMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGS GTDFTLKISRVEAEDLGVYYC FQGSHFPRT FGGGTKLEIK (SEQ ID NO:289) In one embodiment, the antibody comprises the light chain CDRs 1 to 3 (LCDRs 1 to 3) of the anti-MDR1 antibody V6, which comprises a VL chain having the following structure: LCDR1 has the amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO:290) LCDR2 contains amino Acid sequence KVSNRFS (SEQ ID NO:12) LCDR3 contains the amino acid FQGSHFPRT (SEQ ID NO:42) Including nothing.

[0114] In certain embodiments, the antigen-binding site of the first VH chain comprises the heavy chain CDR 1 of an antibody listed in Table 2. Includes ~3 (HCDR 1~3).

[0115] In certain embodiments, the first VH chain comprises HCDRs 1 to 3 of an anti-MDR1 antibody other than an antibody listed in Table 2. 3, and optionally the first VH chain comprises the amino acid sequence of the VH chain of an anti-MDR1 antibody other than an antibody listed in Table 2. Contains arrays.

[0116] In some embodiments, the anti-MDR1 antibody has the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:291) (where X 2 is N, Q or S); AVQLQQSGPELVKTGASVKISCKASGYSFSNYYIHWVKQSHGKSLEWIGFISCYNGATFYNQKFKGKATFTVDTSSSTAY MKFNSLTFEDSAVYYCARLPIQFGNFYPMDYWGQGTSVTVSS (SEQ ID NO:288); or EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLY LQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA (SEQ ID NO:286) The VH chain comprises:

[0117] In some embodiments, the anti-MDR1 antibody has the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:292) wherein X comprises a VH chain having 2 is N, Q or S, and optionally X 2 is Q or S.

[0118] The VL chain(s) can comprise LCDRs 1-3 of the B1-28 antibody as set forth in Table 2, and optionally the V The L chain comprises the amino acid sequence of the VL chain of the B1-28 antibody listed in Table 2.

[0119] In certain embodiments, the VL chain comprises LCDRs 1-3 of an anti-MDR1 antibody other than an antibody listed in Table 2. and optionally, the variable light chain comprises the amino acid sequence of a VL chain of an antibody other than an antibody listed in Table 2. nothing.

[0120] In certain embodiments, the anti-MDR1 antibody comprises the following light chain amino acid sequence: DVLMTQTPVSLSVS LGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCF QASHFPRTFGGGTKLEIK (SEQ ID NO:283); DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:284); or DVVMTQTPRSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHIPPWTFGGGTKLDIK (SEQ ID NO:285).

[0121] In some embodiments, the bispecific antibody binds to MDR1 and a TAA and has an IC50 of 5 or less for a chemotherapeutic agent. by a factor of 1 or more, and a common light chain, VH chain containing HCDRs from the VH chains listed in Table 2. The common light chain comprises a VL chain listed in Table 2. (e.g., the VL of the antibody in Table 2 from which the VH HCDR is derived), or MRK16, 15D 3, or the LCDR from another anti-MDR1 antibody, such as UIC2.

[0122] In some embodiments, the bispecific antibody inhibits the sensitivity of cancer cells to treatment with a chemotherapeutic agent. and increasing the half-maximal inhibitory concentration of the chemotherapeutic agent when co-administered with the antibody. The IC50 is A VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO:293); and a VL chain having the sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:284) at least 5-fold lower than the IC50 of the chemotherapeutic agent when co-administered with an anti-MDR1 antibody containing For example, 6 times lower, 10 times lower, 15 times lower, or 20 times lower).

[0123] In certain embodiments, the second VH chain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:294) and an anti-CD47 antibody comprising HCDRs 1 to 3 of the VH chain of 5F9.

[0124] In certain embodiments, the second VH chain has the following amino acid sequence: EVQLVESGGDLVKPGGSLKLSCAASGTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNTLY LQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSS (SEQ ID NO:295) and an anti-CD47 antibody comprising HCDRs 1 to 3 of the VH chain of B6H12.

[0125] The B6H12 antibody is a murine antibody that binds to human CD47 and has the following amino acid sequence: DIVMTQSPATLSVTPGDRVSLSCRASQTISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEP EDVGVYYCQNGHGFPRTFGGGTKLEIK (SEQ ID NO:296) The VL region comprises:

[0126] The humanized version of the B6H12 antibody has the VH and VL regions as shown below: : B6H12 humanized VH sequence: JPEG2026041756000020.jpg23164HCDR 1 to 3 are underlined and shown in bold. B6H12 humanized VL sequence: JPEG2026041756000021.jpg17164LCDR 1 to 3 are underlined and shown in bold.

[0127] In some embodiments, the bispecific antibody binds to MDR1 and a TAA and has an IC50 of 5 or less for a chemotherapeutic agent. The IL-16A1 and IL-16B1 sequences were compared with those of the other anti-MDR1 antibodies (Table 2), which were compared with the other anti-MDR1 antibodies (Table 2). MRK16, 15D3, or UIC2) or a VH chain listed in Table 2 (e.g., a VH chain containing an HCDR from the VH of an antibody in Table 2 from which the VL LCDR is derived, and The anti-TAA antibody may be an anti-CD47 antibody, such as the murine antibody 5F9. Alternatively, it may be the murine antibody B6H13 or a humanized version thereof. Antibodies include: HCDRs 1 to 3 of the VH region of anti-CD47 antibody 5F9, wherein the VH region comprises the following amino acid sequence: of: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:294); or HCDRs 1 to 3 of the VH region of the anti-CD47 antibody B6H12, wherein the VH region comprises the following amino acid sequence: thing: EVQLVESGGDLVKPGGSLKLSCAASGTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNTLY LQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSS (SEQ ID NO:295). In a specific embodiment, the VH region of the B6H12 antibody comprises an HCDR comprising the sequence GYGMS (SEQ ID NO: 379). 1; HCDR2 containing the sequence TITSGGTYTYYPDSVKG (SEQ ID NO: 299); and HCDR3 containing the sequence SLAGNAMDY (SEQ ID NO: 300). No. 300) containing HCDR3.

[0128] In certain embodiments, the bispecific antibody is the anti-CD47 murine antibody B6H12 or a humanized version thereof. wherein the VH region of the B6H12 antibody comprises the following amino acid sequence: EVQLVESGGDLVKPGGSLKLSCAASGTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNTLY LQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSS (SEQ ID NO:295).

[0129] In certain embodiments, the bispecific antibody is a humanized version of the anti-CD47 murine antibody B6H12. wherein the VH region comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGTFSGYGMSWVRQAPGKGLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCARSLAGNAMDYWGQGTMVTVSS (SEQ ID NO:297).

[0130] In one embodiment the bispecific antibody comprises the following VH region: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO:293) an MDR1-binding domain comprising an HCDR of a VH region from an anti-MDR1 antibody having A TAA binding domain, optionally comprising the following VH region: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:294) a CD47-binding domain comprising an HCDR of a VH region from an anti-CD47 antibody having and A common light chain comprising an LCDR from a VL chain listed in Table 2 (e.g., antibodies B1-28, B1-178, B1-28, B1- 184, B1-188, B1-193B, B1-197, B1-198, B1-201, B1-207, B1-223, B1-85, B1-99, B1-2 7, B1-39, or B1-30).

[0131] In certain embodiments, the bispecific antibody has the following HCDR sequences: HCDR1: RYTMS (SEQ ID NO: 1) ID NO:301), HCDR2: TISSGGGNTYYPDSVKG (SEQ ID NO:302), TISSGGGQTYYPDSVKG (SEQ ID NO:303), or TISSGGGSTYYPDSVKG (SEQ ID NO:304), and HCDR3: YGAGDAWFAY (SEQ I HCDRs 1 to 3 of the VH region derived from an anti-MDR1 antibody having the following HCDR sequence: R1: NYNMH (SEQ ID NO:306), HCDR2: TIYPGNDDTSYNQKFKD (SEQ ID NO:307), and HCDR3 HCDR of the VH region from an anti-CD47 antibody having GGYRAMDY (SEQ ID NO:308) and an anti-MDR1 antibody B1- and a common light chain comprising the LCDR of the 28 VL region. In certain embodiments, the LCDR sequence is 1: RSSQNIVHSTGNTYLD (SEQ ID NO:11), LCDR2: KVSNRFS (SEQ ID NO:12), and LCDR3: FQGSHIPRT (SEQ ID NO:13).

[0132] Unless otherwise specified, CDRs are defined according to Kabat nomenclature.

[0133] In certain embodiments, the first VH chain of the bispecific antibody molecule comprises: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:291) where X 2 is N, Q or S, and the second VH chain comprises an amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:294) and / or the two VL chains comprise an amino acid sequence comprising the amino acid sequence of the B1-28 antibody described in Table 2. It comprises an amino acid sequence comprising the VL chain of

[0134] In certain embodiments, the bispecific antibody comprises a VH chain comprising HCDRs 1-3 from the VH chain of B1.188; A common VL chain containing LCDRs 1 to 3 from the VL chain of the anti-MDR1 antibody MRK16, and a VH chain of the anti-CD47 antibody 5F9 HCDRs 1-3 from the VH chain of B1.188, HCDRs 1-3 from the VL chain of MRK16. CDRs 1-3, and HCDRs 1-3 from the VH chain of 5F9 are listed in Table 5. In certain embodiments, The bispecific antibody contains the VH chain of B1.188, the VL chain of MRK16 as the common light chain, and the VH chain of 5F9.

[0135] In certain embodiments, the bispecific antibody comprises a VH chain comprising HCDRs 1-3 from the VH chain of B1.261; A common VL chain containing LCDRs 1-3 from the VL chain of the anti-MDR1 antibody, MRK16, and the VL chain of the anti-CD47 antibody, 5F9 It contains a VH chain containing HCDRs 1 to 3 from the H chain. It contains HCDRs 1 to 3 from the VH chain of B1.261 and HCDRs 1 to 3 from the VL chain of MRK16. and HCDRs 1-3 from the VH chain of 5F9 are set forth in Table 5. The bispecific antibody contains the VH chain of B1.261, the VL chain of MRK16 as a common light chain, and the VH chain of 5F9. .

[0136] In certain embodiments, the bispecific antibody comprises a VH comprising HCDRs 1-3 from the VH chain of B1.129. a common VL chain containing LCDRs 1-3 from the VL chain of the anti-MDR1 antibody, MRK16, and an anti-CD47 antibody, 5F VH chains containing HCDRs 1-3 from the VH chain of B1.129 and the VL chain of MRK16. LCDRs 1-3 from the VH chain of 5F9 and HCDRs 1-3 from the VH chain of 5F9 are set forth in Table 5. The bispecific antibody contains the VH chain of B1.129, the VL chain of MRK16 as a common light chain, and the VH chain of 5F9. include.

[0137] In certain embodiments, the bispecific antibody comprises a VH chain comprising HCDRs 1-3 from the VH chain of B1.225; A common VL chain containing LCDRs 1-3 from the VL chain of the anti-MDR1 antibody, MRK16, and the VL chain of the anti-CD47 antibody, 5F9 It contains a VH chain containing HCDRs 1 to 3 from the H chain. It contains HCDRs 1 to 3 from the VH chain of B1.225 and HCDRs 1 to 3 from the VL chain of MRK16. and HCDRs 1-3 from the VH chain of 5F9 are set forth in Table 5. The bispecific antibody contains the VH chain of B1.225, the VL chain of MRK16 as a common light chain, and the VH chain of 5F9. .

[0138] In certain embodiments, the bispecific antibody comprises a VH chain comprising HCDRs 1-3 from the VH chain of B1.223; A common VL chain containing LCDRs 1-3 from the VL chain of the anti-MDR1 antibody, MRK16, and the VL chain of the anti-CD47 antibody, 5F9 It contains a VH chain containing HCDRs 1 to 3 from the H chain. It contains HCDRs 1 to 3 from the VH chain of B1.223 and HCDRs 1 to 3 from the VL chain of MRK16. and HCDRs 1-3 from the VH chain of 5F9 are set forth in Table 5. The bispecific antibody contains the VH chain of B1.223, the VL chain of MRK16 as a common light chain, and the VH chain of 5F9. .

[0139] In certain embodiments, the bispecific antibody comprises a VH comprising HCDRs 1-3 from the VH chain of B1.28. a common VL chain containing LCDRs 1-3 from the VL chain of B1.28, and a VH chain from the anti-CD47 antibody, 5F9 HCDRs 1 to 3 from the VH chain of B1.28, LCDR 1 to 3 from the VL chain of B1.28 HCDRs 1-3 from the VH chain of 5F9 are listed in Table 5. In certain embodiments, the double specific The heterologous antibody contains the VH chain of B1.28, the VL chain of B1.28 as the common light chain, and the VH chain of 5F9. In this embodiment, the VH chain of B1.28, the VL chain of B1.28, and the VH chain of 5F9 are humanized and shown in Table 5. It may have the sequence described.

[0140] In a particular example, the first anti-MDR1 and anti-CD47 bispecific antibody of the present disclosure has the following sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the 15D3 antibody; a second VH chain comprising HCDRs 1 to 3 of the VH chain of the 5F9 antibody; and LCDR1 to LCDR3 of the VL chain of the B1.27 antibody, B1.VL6.CDR3 mod antibody, B1.225v1 antibody, or B1.89v antibody The common VL chain contains 3. The LCDRs 1 to 3 of the B1.VL6.CDR3 mod antibody, B1.225v1 antibody, and B1.89v antibody The sequences of the VL chain and VL chain are listed in Table 5.

[0141] In a particular example, the second anti-MDR1 and anti-CD47 bispecific antibody of the disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.188 antibody; a second VH chain comprising HCDRs 1 to 3 of the VH chain of the 5F9 antibody; and A common VL chain containing LCDRs 1 to 3 of the VL chain of the MRK16 antibody.

[0142] In a particular example, the third anti-MDR1 and anti-CD47 bispecific antibody of the present disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.225 antibody; a second VH chain comprising HCDRs 1 to 3 of the VH chain of the 5F9 antibody; and A common VL chain containing LCDRs 1 to 3 of the VL chain of the MRK16 antibody.

[0143] In a particular example, the fourth anti-MDR1 and anti-CD47 bispecific antibody of the disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.28 antibody; a second VH chain comprising HCDRs 1 to 3 of the VH chain of the 5F9 antibody; and A common VL chain containing LCDRs 1 to 3 of the VL chain of the B1.28 antibody.

[0144] In a particular example, the first VH chain can comprise the amino acid sequence of the humanized VH chain of B1.28.

[0145] In a particular example, the fifth anti-MDR1 and anti-CD47 bispecific antibody of the present disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.261 antibody; a second VH chain comprising HCDRs 1 to 3 of the VH chain of the 5F9 antibody; and A common VL chain containing LCDRs 1 to 3 of the VL chain of the MRK16 antibody.

[0146] In a particular example, the first VH chain can be a humanized VH chain of the B1.261 antibody. In particular, the humanized version can comprise the amino acid sequence of the VH of the B1.261 VH4 antibody. In certain examples, the common VL chain may be a humanized VL chain of the MRK16 antibody.

[0147] In a particular example, the sixth anti-MDR1 and anti-CD47 bispecific antibody of the present disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.261 antibody; a second VH chain comprising HCDRs 1 to 3 of the VH chain of the 5F9 antibody; and A common VL chain containing LCDRs 1 to 3 of the VL chain of the B1.261 antibody.

[0148] In a particular example, the first VH chain can be a humanized VH chain of the B1.261 antibody. In particular, the humanized version can comprise the amino acid sequence of the VH of the B1.261 VH4 antibody. In certain instances, the common VL chain may be the humanized VL chain of the B1.261 antibody. , the humanized version may comprise the amino acid sequence of the VL of the B1.261 VH4 antibody.

[0149] The following bispecific antibodies target human and cynomolgus CD47 and human ABCB on the cell surface: 1 may be particularly useful for binding to: 15D3DD KT14KK B1.28.huL2 LC; 15D3DD KT14KK B1VL6 / CDR3v2 LC; 15D3DD KT14KK MRK16v5 LC; and 15D3DD KT14KK B1.89v1 LC (Figure 17A -17B). The amino acid sequences of the heavy and light chains of these antibodies are as follows:

[0150] 15D3DD heavy chain: JPEG2026041756000022.jpg55164

[0151] KT14KK heavy chain: JPEG2026041756000023.jpg55164

[0152] In the heavy chain sequence above, the VH region is shown in bold. The HCDRs are italicized and underlined. The bispecific antibodies encompassed by the present invention may comprise any of these heavy chains or any of these heavy chains. It may contain only the HCDRs or only the VH chain present in

[0153] B1.28.huL2 common light chain: JPEG2026041756000024.jpg19163

[0154] B1VL6 / CDR3v2 common light chain: JPEG2026041756000025.jpg17161

[0155] MRK16v5 LC common light chain: JPEG2026041756000026.jpg16161

[0156] B1.89v1 common light chain: JPEG2026041756000027.jpg17161

[0157] The LCDR is shown in bold. Bispecific antibodies encompassed by the present invention have these common light chains. one in combination with the 15D3DD heavy chain and KT14 heavy chain or its HCDR or VH chain. It can be done.

[0158] In particular examples, the TAA is Her2 or PD-L1.

[0159] [Anti-MDR1 and anti-PD-L1 bispecific antibody] In certain embodiments, the TAA can be programmed cell death ligand 1 (PD-L1). Also known as cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7-H1) In one embodiment, the bispecific antibody molecule that binds to MDR1 and PD-L1 is As described in the previous section, the first VH chain comprises a common light chain and a first VH chain, and the second VH chain comprises: The amino acid sequence below: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 314) and VH chain HCDRs 1 to 3, including:

[0160] HCDRs 1 to 3 defined according to the Kabat nomenclature are as follows: HCDR1: DSWIH (SEQ ID NO: 315) HCDR2: WISPYGGSTYYADSVKG (SEQ ID NO: 316) HCDR3: RHWPGGFDY (SEQ ID NO: 317)

[0161] The second VH chain of the bispecific antibody that binds to MDR1 and PD-L1 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 314) an amino acid sequence having at least 80%, at least 90%, at least 95%, or 100% identity to It may have an acid sequence.

[0162] The second VH chain of the bispecific antibody that binds to MDR1 and PD-L1 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 318) an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to The amino acid sequence may be present in the heavy chain.

[0163] In a particular example, the anti-MDR1 and anti-PDL1 bispecific antibody of the present disclosure comprises the following sequence: : a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.261 antibody; a second VH chain comprising HCDRs 1-3 of the VH chain of the atezolizumab antibody; and A common VL chain comprising LCDRs 1 to 3 of the VL chain of the B1.261 antibody or the MRK16 antibody.

[0164] In certain examples, the first VH chain of the anti-MDR1 and anti-PD-L1 bispecific antibody is humanized, It contains the following amino acid sequence: B1.261.huH1(VH4): EVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSALKSRLTISKDNSKNQVSL KLSSVTAADTAVYYCARHGRWLLQRGGAMDYWGQGTMVTVSS (SEQ ID NO:126) B1.261.huH2(VH3): EVQLVESGGGLIQPGGSLRLSCAVSGFSLTSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSALKSRLTISKDNSKNTVYL QMNSLRAEDTAVYYCARHGRWLLQRGGAMDYWGQGTMVTVSS (SEQ ID NO:129)

[0165] In a particular example, the common VL chain of the anti-MDR1 and anti-PDL1 bispecific antibody is humanized and has the following structure: Amino acid sequence includes: JPEG2026041756000028.jpg28160

[0166] [Anti-MDR1 and anti-HER2 bispecific antibody] In certain embodiments, the TAA can be HER2, Erb-B2 receptor tyrosine kinase 2, or HER2 is a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. The HER2 protein does not have a ligand-binding domain itself, so it cannot bind to growth factors. However, it does bind to other ligand-binding EGF receptor family members. They bind to each other to form heterodimers, stabilizing ligand binding and promoting downstream signaling pathways. Enhances kinase-mediated activation. Amplification and / or overexpression of the gene encoding HER2 has been reported in a number of cancers, including breast and ovarian cancer.

[0167] In certain embodiments, the bispecific antibody molecule that binds to MDR1 and HER2 is The first VH chain comprises a common light chain and a first VH chain as described in section 1. The second VH chain comprises a common light chain and a first VH chain as described in section 1. It contains HCDRs 1 to 3 of the VH chain of an anti-Her2 antibody such as rutuzumab.

[0168] Trastuzumab heavy chain sequence: evqlvesggglvqpggslrlscaasgfnikdtyihwvrqapgkglewvariyptngytryadsvkgrftisadtskntay lqmnslraedtavyycsrwggdgfyamdywgqgtlvtvss astkgpsvfplapsskstsggtaalgclvkdyfpepvtvs wnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellgg psvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngk eykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppv ldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk (SEQ ID NO:320) The VH region is underlined.

[0169] HCDRs 1 to 3, defined according to the Kabat nomenclature, are as follows: HCDR1: GFNIKDT (SEQ ID NO:321) HCDR2: YPTNGY (SEQ ID NO:322) HCDR3: WGGDGFYAMDY (SEQ ID NO:323)

[0170] The second VH chain of the bispecific antibody that binds MDR1 and HER2 may be the VH chain of trastuzumab or The amino acid sequence of the heavy chain of trastuzumab is at least 80%, at least 90%, or at least The amino acid sequences may be 95% or 100% identical to each other.

[0171] Pertuzumab heavy chain sequence: evqlvesggglvqpggslrlscaasgftftdytmdwvrqapgkglewvadvnpnsggsiynqrfkgrftlsvdrskntly lqmnslraedtavyycarnlgpsfyfdywgqgtlvtvss astkgpsvfplapsskstsggtaalgclvkdyfpepvtvsw nsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggp svflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngke ykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvl dsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg (SEQ ID NO:324) The VH region is underlined.

[0172] HCDRs 1 to 3, defined according to the Kabat nomenclature, are as follows: HCDR1: GFTFTDY (SEQ ID NO:325) HCDR2: NPNSGG (SEQ ID NO:326) HCDR3: NLGPSFYFDY (SEQ ID NO:327)

[0173] The second VH chain of the bispecific antibody that binds MDR1 and HER2 may be the VH chain of pertuzumab or At least 80%, at least 90%, at least The amino acid sequence may have 95% or 100% identity.

[0174] In a particular example, the first anti-MDR1 and anti-HER2 bispecific antibody of the present disclosure has the following sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.129 antibody; a second VH chain comprising HCDRs 1-3 of the VH chain of pertuzumab or a pertuzumab antibody; and A common VL chain containing LCDRs 1 to 3 of the VL chain of the B1.129 antibody.

[0175] In a particular example, the second anti-MDR1 and anti-HER2 bispecific antibody of the disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.188 antibody; a second VH chain comprising HCDRs 1-3 of the VH chain of pertuzumab or a pertuzumab antibody; and A common VL chain comprising LCDRs 1 to 3 of the VL chain of the B1.188 antibody or the MRK16 antibody.

[0176] In a particular example, the third anti-MDR1 and anti-HER2 bispecific antibody of the present disclosure has the sequence: Including: a first VH chain comprising HCDRs 1 to 3 of the VH chain of the B1.261 antibody; a second VH chain comprising HCDRs 1-3 of the VH chain of pertuzumab or a pertuzumab antibody; and A common VL chain comprising LCDRs 1 to 3 of the VL chain of the B1.261 antibody or the MRK16 antibody.

[0177] In a particular example, the first VH chain of the anti-MDR1 and anti-PDL1 bispecific antibody is humanized, It contains the following amino acid sequence: B1.261.huH1(VH4): EVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSALKSRLTISKDNSKNQVSL KLSSVTAADTAVYYCARHGRWLLQRGGAMDYWGQGTMVTVSS (SEQ ID NO:126) B1.261.huH2(VH3): EVQLVESGGGLIQPGGSLRLSCAVSGFSLTSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSALKSRLTISKDNSKNTVYL QMNSLRAEDTAVYYCARHGRWLLQRGGAMDYWGQGTMVTVSS (SEQ ID NO:129)

[0178] In certain instances, the common VL chain of the anti-MDR1 and anti-PDL1 bispecific antibody is humanized and is Contains the amino acid sequence: JPEG2026041756000029.jpg29161

[0179] As described elsewhere herein, among a VL chain, a first VH chain, and a second VH At least one, two, or all of the Fc regions of the VH chain can be humanized. The VH chain may contain substitutions that increase heterodimerization between the first and second VH chains. In such a case, the first heavy chain can be humanized to include charge pair substitutions K392D and K409D, and the second The heavy chain of may contain the charge pair substitutions E356K and D399K.

[0180] In certain embodiments, one or more of the VH and VL chains are, e.g., at least one, two, ... , replacing three or four framework regions with framework regions from a human antibody. The polypeptide can be humanized by

[0181] In certain embodiments, the bispecific antibody is directed against cancer cells that express both MDR1 and a tumor-associated antigen. In certain embodiments, the bispecific antibody specifically binds to MDR1 or tumor-associated antigen. The binding of the IgG4-dependent agonist to the IgG4-dependent agonist expressed both MDR1 and a tumor-associated antigen was compared to that of the IgG4-dependent agonist to .... It preferentially binds to cancer cells that express it.

[0182] In certain embodiments, the bispecific antibody is directed against cancers that express both MDR1 and tumor-associated antigens. Binds to cells but has low or undetectable expression of MDR1 or related tumor-associated antigens It does not bind (or binds with significantly lower affinity) to normal cells. The bispecific antibody shown does not bind to cells that do not express both MDR1 and TAA.

[0183] In certain embodiments, the multispecific antibodies of the present disclosure may comprise a common light chain. When used, the term "common light chain" generally refers to the same light chain in a multispecific antibody. This refers to the use and incorporation of two copies of a single strand. In other words, the constructed multispecific In the antibody, the light chain associates with an MDR1-specific heavy chain, and a second copy of that same light chain binds to the tumor. In some embodiments, the common light chain is associated with the LCD of a VL chain listed in Table 2. The VH chain may comprise the HCDRs of a VH chain listed in Table 2.

[0184] Bispecific antibodies encompassed by the present disclosure bind to MDR-1 and CD47 and bind to the 15D3 antibody HCDR from the VH chain of the 5F9 antibody, and LCDR from the VL chain of the MRK16 antibody. Specifically, the bispecific antibodies of the present disclosure include the following combinations: Not at all. The following VH sequences: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO:293) a VH chain with an HCDR present in The following VH sequences: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:294) a VH chain having an HCDR present in The following VL sequences: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO:283) The common variable light chain has an LCDR present in

[0185] Specifically, the bispecific antibodies of the present disclosure do not include the following combinations: The following HCDR sequences: HCDR1: RYTMS (SEQ ID NO:301), HCDR2: TISSGGGNTYYPDSVKG (SEQ ID NO: NO:302), and a VH region from an anti-MDR1 antibody with HCDR3: YGAGDAWFAY (SEQ ID NO:305). HCDR; The following HCDR sequences: HCDR1: NYNMH (SEQ ID NO:306), HCDR2: TIYPGNDDTSYNQKFKD (SEQ ID NO: H of the VH region from an anti-CD47 antibody with HCDR3: GGYRAMDY (SEQ ID NO:308) CDRs; and The following LCDR sequences: LCDR1: RSSQSIVHSTGNTYLE (SEQ ID NO:25), LCDR2: KISNRFS (SEQ ID NO: NO:328), and LCDR3 of an anti-MDR1 antibody having LCDR4: FQASHFPRT (SEQ ID NO:329). Common light chain.

[0186] Certain bispecific antibodies that bind to MDR1 and CD47 on the cell surface share an anti-MDR1 light chain as a common light chain. R1 antibody, and may contain LCDRs 1 to 3 of the VL chain of V6; anti-MDR1 antibodies B1.28, B1.30, B1.89, B1.129, B1.2 HCDRs 1 to 3 of the VH chain of 25, B1.261, 15D3, or MRK16, and the VH of the 5F9 antibody or B6H12 antibody Certain bispecific antibodies that bind to MDR1 and CD47 on the cell surface may contain HCDRs 1-3 of the IgG1 chain. The body may comprise an anti-MDR1 antibody as a common light chain or a humanized version thereof, a VL chain of V6 or a human and the anti-MDR1 antibodies B1.28, B1.30, B1.89, B1.129, B1.225, B1.261, 15D 3, or the VH chain of MRK16 or a humanized version thereof and the VH chain of the 5F9 antibody or the B6H12 antibody or the VH chain of a humanized version of the B6H12 or 5F9 antibodies.

[0187] In certain embodiments, the subject antibodies are recombinant or modified antibodies, e.g., chimeric, Humanized, deimmunized, or in vitro generated antibodies. The term "recombinant" or "modified" antibody refers to an antibody that is prepared, expressed, or produced by recombinant means. The term "antibody" is intended to include all antibodies that have been transfected or isolated, for example, (i) by transfection into host cells; (ii) an antibody expressed using a recombinant expression vector transfected with the antibody; (iii) antibodies isolated from a clinical antibody library; or (iv) an antibody isolated from an animal (e.g., a mouse) transgenic for the antibody; or By other means, including splicing human immunoglobulin gene sequences into other DNA sequences. The recombinant antibodies include prepared, expressed, produced or isolated antibodies. Such recombinant antibodies include humanized antibodies, CDR-grafted antibodies, chimeric antibodies, deimmunized antibodies, and in vitro generated antibodies are included. The antibodies may optionally include constant regions derived from human germline immunoglobulin sequences.

[0188] The modified antibodies may contain modified domains, and any antibody domain may be a naturally occurring In some embodiments, modified antibodies include those that can be modified from their existing form. a modified CH2 and / or CH3 domain, and a modified Fc domain. In some instances, the modified Fc domain may comprise an altered heavy chain. The ring effect can be utilized, as described, for example, in Gunasekeran et al. (2010) Journal of Biology Cal Chemistry 285, 19637-19646, the disclosure of which is incorporated herein by reference in its entirety. including, but not limited to, through the use of procedures described in In some instances, bispecific antibodies assemble via charge pair substitutions in the CH3 domain. For example, one heavy chain is engineered to contain K392D and K409D substitutions and the other and one heavy chain modified to contain E356K and D399K substitutions. No. Charge-pair substituted chains preferentially form heterodimers with each other. Numbering of amino acid substitutions The numbers are according to the EU numbering system for Ig HC.

[0189] In some examples, the antibodies of the present disclosure comprise charge pair substitutions. The antibody does not contain charge pair substitutions. In some instances, the preferred heterodimeric form of the desired chains Alternative means of facilitating synthesis may be used.

[0190] In some cases, the modified heavy chain comprises a knob-into-hole modification. "Knob-into-hole" amino acid engineering is a rational design strategy in antibody engineering. Yes, used for heavy chain heterodimerization and production of multispecific antibodies (including bispecific IgG antibodies) For example, bispecific antibodies made from two monoclonal antibodies with different specificities are used. Incorporating the knobs-into-holes strategy into heterologous antibodies, monoclonal antibody 1 (mAb1) The “knob” is formed on the CH3 of the heavy chain of monoclonal antibody 2 (mAb2), and the “knob” is formed on the CH3 of the heavy chain of monoclonal antibody 2 (mAb2). The knobs are used to manipulate amino acid changes to form a "knob." The bases can be represented by large amino acids such as threonine (T), and the holes can be represented by small amino acids such as threonine (T). For example, a knob-into-hole pair modification can be a T22Y substitution in the first CH3 domain. and a Y86T substitution in the partner CH3 domain. Examples of two-hole modifications are described in Carter, J. Immunol. Methods, 248(1-2):7-15 (2001); Ridgway, JB et al. Protein Eng. 9(7):617-2 (1996); and Merchant, AM et al. Nat. Bi 16(7):677-81 (1998), the disclosures of which are incorporated herein in their entirety. In antibodies generated from paired knob-into-hole engineered domains, , bispecific heterodimers will generally represent the major fraction.

[0191] As described above, the anti-MDR1 antibodies of the present invention specifically bind to one or more epitopes of MDR1. Therefore, the epitope is an MDR1 epitope. The size of the R1 epitope can vary, and the MDR1 epitope can be, for example, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 4 aa~10 aa, 5 aa~10 aa, 6 aa~10 aa, 4 aa~8 3 aa or less to 12 aa or more, including but not limited to aa, 5 aa to 8 aa, 6 aa to 8 aa, etc. and polypeptides having a continuous stretch of the MDR1 sequence, which may range from This includes cases where

[0192] In some embodiments, MDR1 epitopes include, but are not limited to, at least about 75%, at least about 80% relative to a contiguous stretch of the MDR1 sequence that is not defined , at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least It may be formed by a polypeptide having about 99%, or 100%, amino acid sequence identity: Human MDR1 sequence MDLEGDRNGGAKKKNFFKLNNKSEKDKKEKKPTVSVFSMFRYSNWLDKLYMVVGTLAAIIHGAGLPLMMLVFGEMTDIFA NAGNLEDLMSNITNRSDINDTGFFMNLEEDMTRYAYYYSGIGAGVLVAAYIQVSFWCLAAGRQIHKIRKQFFHAIMRQEI GWFDVHDVGELNTRLTDDVSKINEGIGDKIGMFFQSMATFFTGFIVGFTRGWKLTLVILAISPVLGLSAAVWAKILSSFT DKELLAYAKAGAVAEEVLAAIRTVIAFGGQKKELERYNKNLEEAKRIGIKKAITANISIGAAFLLIYASYALAFWYGTTL VLSGEYSIGQVLTVFFSVLIGAFSVGQASPSIEAFANARGAAYEIFKIIDNKPSIDSYSKSGHKPDNIKGNLEFRNVHFS YPSRKEVKILKGLNLKVQSGQTVALVGNSGCGKSTTVQLMQRLYDPTEGMVSVDGQDIRTINVRFLREIIGVVSQEPVLF ATTIAENIRYGRENVTMDEIEKAVKEANAYDFIMKLPHKFDTLVGERGAQLSGGQKQRIAIARALVRNPKILLLDEATSA LDTESEAVVQVALDKARKGRTTIVIAHRLSTVRNADVIAGFDDGVIVEKGNHDELMKEKGIYFKLVTMQTAGNEVELENA ADESKSEIDALEMSSNDSRSSLIRKRSTRRSVRGSQAQDRKLSTKEALDESIPVSFWRIMKLNLTEWPYFVVGVFCAII NGGLQPAFAIIFSKIIGVFTRIDDPETKRQNSNLFSLLFLALGIISFITFFLQGFTFGKAGEILTKRLRYMVFRSMLRQD VSWFDDPKNTTGALTTRLANDAAQVKGAIGSRLAVITQNIANLGTGIIISFIYGWQLTLLLLAIVPIIAIAGVVEMKMLS GQALKDKKELEGSGKIATEAIENFRTVVSLTQEQKFEMHYAQSLQVPYRNSLRKAHIFGITFSFTQAMMYFSYAGCFRFG AYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSFAPDYAKAKISAAHIIMIIEKTPLIDSYSTEGLMPNTLEGNVTFGEV VFNYPTRPDIPVLQGLSLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPLAGKVLLDGKEIKRLNVQWLRAHLGIVSQEP ILFDCSIAENIAYGDNSRVVSQEEIVRAAKEANIHAFIESLPNKYSTKVGDKGTQLSGGQKQRIAIARALVRQPHILLLD EATSALDTESEKVVQEALDKAREGRTCIVIAHRLSTIQNADLIVVFQNGRVKEHGTHQQLLAQKGIYFSMVSVQAGTKRQ (SEQ ID NO: 330); or a rodent MDR1 sequence, such as the following mouse MDR1 sequence or mouse MDR1b sequence: Mouse MDR1a sequence: MELEEDLKGRADKNFSKMGKKSKKEKKEKKPAVSVLTMFRYAGWLDRLYMLVGTLAAIIHGVALPLMMLIFGDMTDSFAS VGNVSKNSTNMSEADKRAMFAKLEEEMTTYAYYYTGIGAGVLIVAYIQVSFWCLAAGRQIHKIRQKFFHAIMNQEIGWFD VHDVGELNTRLTDDVSKINEGIGDKIGMFFQAMATFFGGFIIGFTRGWKLTLVILAISPVLGLSAGIWAKILSSFTDKEL HAYAKAGAVAEEVLAAIRTVIAFGGQKKELERYNNNLEEAKRLGIKKAITANISMMGAAFLLIYASYALAFWYGTSLVISK EYSIGQVLTVFFSVLIGAFSVGQASPNIEAFANARGAAYEVFKIIDNKPSIDSFSKSGHKPDNIQGNLEFKNIHFSYPSR KEVQILKGLNLKVKSGQTVALVGNSGCGKSTTVQLMQRLYDPLDGMVSIDGQDIRTINVRYLREIIGVVSQEPVLFATTI AENIRYGREDVTMDEIEKAVKEANAYDFIMKLPHQFDTLVGERGAQLSGGQKQRIAIARALVRNPKILLLDEATSALDTE SEAVVQAALDKAREGRTTIVIAHRLSTVRNADVIAGFDGGVIVEQGNHDELMREKGIYFKLVMTQTAGNEIELGNEACKS KDEIDNLDMSSKDSGSSLIRRRSTRKSICGPHDQDRKLSTKEALDEDVPPASFWRILKLNSTEWPYFVVGIFCAIINGGL QPAFSVIFSKVVGVFTNGGPPETQRQNSNLFSLLFLILGIISFITFFLQGFTFGKAGEILTKRLRYMVFKSMLRQDVSWF DDPKNTTGALTTRLANDAAQVKGATGSRLAVIFQNIANLGTGIIISLIYGWQLTLLLLAIVPIIAIAGVVEMKMLSGQAL KDKKELEGSGKIATEAIENFRTVVSLTREQKFETMYAQSLQIPYRNAMKKAHVFGITFSFTQAMMYFSYAACFRFGAYLV TQQLMTFENVLLVFSAIVFGAMAVGQVSSFAPDYAKATVSASHIIRIIEKTPEIDSYSTQGLKPNMLEGNVQFSGVVFNY PTRPSIPVLQGLSLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPMAGSVFLDGKEIKQLNVQWLRAQLGIVSQEPILFD CSIAENIAYGDNSRVVSYEEIVRAAKEANIHQFIDSLPDKYNTRVGDKGTQLSGGQKQRIAIARALVRQPHILLLDEATS ALDTESEKVVQEALDKAREGRTCIVIAHRLSTIQNADLIVVIQNGKVKEHGTHQQLLAQKGIIFSMVSVQAGAKRS (SE Q ID NO:331) MouseMDR1b sequence: MEFEENLKGRADKNFSKMGKKSKKEKKKPAVGVFGMFRYADWLDKLCMILGTLAAIIHGTLLPLLMLVFGNMTDSFTK AEASILPSITNQSGPNSTLIISNSSLEEEMAIYAYYYTGIGAGVLIVAYIQVSLWCLAAGRQIHKIRQKFFHAIMNQEIG WFDVHDVGELNTRLTDDVSKINDGIGDKIGMFFQSITTFLAGFIIGFISGWKLTLVILASPLIGLSSALWAKVLTSFTN KELQAYAKAGAVAEEVLAAIRTVIAFGGQQKELERYNKNLEEAKNVGIKKAITASISIGIAYLLVYASYALAFWYGTSLV LSNEYSIGEVLTVFFSILLGTFSIGHLAPNIEAFANARGAAFEIFKIIDNEPSIDFSTKGYKPDSIMGNLEFKNVHFNY PSRSEVQILKGLNLKVKSGQTVALVGNSGCGKSTTVQLMQRLYDPLEGVVSIDGQDIRTINVRYLREIIGVVSQEPVLFA TTIAENIRYGREDVTMDEIEKAVKEANAYDFIMKLPHQFDTLVGERGAQLSGGQKQRIAIARALVRNPKILLLDEATSAL DTESEAVVQAALDKAREGRTTIVIAHRLSTVRNADVIAGFDGGVIVEQGNHDELMREKGIYFKLVMTQTRGNEIEPGNNA YGSQSDTDASELTSEESKSPLIRRSIYRSVHRKQDQERRLSMKEAVDEDVPLVSFWRILNLNLSEWPYLLVGVLCAVING CIQPVFAIVFSRIVGVFSRDDDHETKRQNCNLFSLFLVMGLISFVTYFFQGFTFGKAGEILTKRVRYMVFKSMLRQDIS WFDDHKNSTGSLTTRLASDASSVKGAMGARLAVVTQNVANLGTGVILSLVYGWQLTLLLVVIIPLIVLGGIIEMKLLSGQ ALKDKKQLEISGKIATEAIENFRTIVSLTREQKFETMYAQSLQVPYRNAMKKAHVFGITFSFTQAMMYFSYAACFRFGAY LVAQQLMTFENVMLVFSAVVFGAMAAGNTSSFAPDYAKAKVSASHIIRIIEKTPEIDSYSTEGLKPTLLEGNVKFNGVQF NYPTRPNIPVLQGLSLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPMAGSVFLDGKEIKQLNVQWLRAHLGIVSQEPIL FDCSIAENIAYGDNSRAVSHEEIVRAAKEANIHQFIDSLPDKYNTRVGDKGTQLSGGQKQRIAIARALVRQPHILLLDEA TSALDTESEKVVQEALDKAREGRTCIVIAHRLSTIQNADLIVVIENGKVKEHGTHQQLLAQKGIYFSMVQAGAKRS (SE Q ID NO:332), or non-human primate sequences, such as Pan troglodytes (chimpanzees) )array: MDLEGDRNGGAKKKNFFKLNNKSEKDKKEKKPTVSVFSMFRYSNWLDKLYMVVGTLAAIIHGAGLPLMMLVFGEMTDIFA NAGNLEDLMSNITNRSDINDTGFFMNLEEDMTRYAYYYSGIGAGVLVAAYIQVSFWCLAAGRQIHKIRKQFFHAIMRQEI GWFDVHDVGELNTRLTDDVSKINEGIGDKIGMFFQSMATFFTGFIVGFTRGWKLTLVILAISPVLGLSAAVWAKILSSFT DKELLAYAKAGAVAEEVLAAIRTVIAFGGQKKELERYNKNLEEAKRIGIKKAITANISIGAAFLLIYASYALAFWYGTTL VLSGEYSIGQVLTVFFSVLIGAFSVGQASPSIEAFANARGAAYEIFKIIDNKPSIDSYSKSGHKPDNIKGNLEFRNVHFS YPSRKQVKILKGLNLKVQSGQTVALVGNSGCGKSTTVQLMQRLYDPTEGMVSVDGQDIRTINVRFLREIIGVVSQEPVLF ATTIAENIRYGRENVTMDEIEKAVKEANAYDFIMKLPHKFDTLVGERGAQLSGGQKQRIAIARALVRNPKILLLDEATSA LDTESEAVVQVALDKARKGRTTIVIAHRLSTVRNADVIAGFDDGVIVEKGNHDELMKEKGIYFKLVTMQTAGNEVELENA ADESKSEIDALEMSSNDSRSSLIRKRSTRRSVRGSQAQDRKLSTKEALDESIPVSFWRIMKLNLTEWPYFVVGVFCAII NGGLQPAFAIIFSKIIGVFTRIDDPETKRQNSNLFSLLFLVLGIISFITFFLQGFTFGKAGEILTKRLRYMVFRSMLRQD VSWFDDPKNTTGALTTRLANDAAQVKGAIGSRLAVITQNIANLGTGIIISFIYGWQLTLLLLAIVPIIAIAGVVEMKMLS GQALKDKKELEGAGKIASEAIENFRTVVSLTQEQKFEMHYAQSLQVPYRNSLRKAHIFGITFSFTQAMMYFSYAGCFRFG AYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSFAPDYAKAKISAAHIIMIIEKTPLIDSYSTEGLTPNTLEGNVTFGEV VFNYPTRPDIPVLQGLSLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPLAGKVLLDGKEIKRLNVQWLRAHLGIVSQEP ILFDCSIAENIAYGDNSRVVSQEEIVRAAKEANIHAFIESLPNKYSTRVGDKGTQLSGGQKQRIAIARALVRQPHILLLD EATSALDTESEKVVQEALDKAREGRTCCIVIAHRLSTIQNADLIVVFQNGRVKEHGTHQQLLAQKGIIFSMVSVQAGTKRQ (SEQ ID NO:333)、or Macaca fascicularis(カニクイザル) sequence: MDLEGDRNGGAEKKNFFKLNNKSKKDKKERKPTVSVFSMFRYSNWLDKLYMVVGTLAAIIHGAGLPLMMLVFGDMTDTFA NAGNLGDLGALLFNNTNSNITDTVPVMNLEEDMTRYAYYYSGIGAGVLVAAYIQVSFWCLAAGRQIHKIRKQFFHAIMR QEIGWFDVHDVGELNTRLTDDVSKINEGIGDKIGMFFQSMATFFTGFIVGFTRGWKLTLVILAISPVLGLSAAVWAKILS SFTDKELLAYAKAGAVAEEVLAAIRTVIAFGGQKKELERYNKNLEEAKRIGIKKAITANISIGAAFLLIYASYALAFWYG TTLVLSKEYSIGQVLTVFFSVLIGAFSVGQASPSIEAFANARGAAFEIFKIIDNKPSIDSYSKSGHKPDNIKGNLEFRNV HFSYPSRKEVKILKGLNLKVQSGQTVALVGNSGCGKSTTVQLMQRLYDPTEGMVSVDGQDIRTINVRFLREIIGVVSQEP VLFATTIAENIRYGREDVTMDEIEKAVKEANAYDFIMKLPQKFDTLVGERGAQLSGGQKQRIAIARALVRNPKILLLDEA TSALDTESEAVVQVALDKARKGRTTIVIAHRLSTVRNADVIAGFDDGVIVEKGNHDELMKEKGIYFKLVTMQTAGNEIEL ENAADESKSEIDTLEMSSHDSGSSLIRKRSTRRSVRGSQGQDRKLSTKEALDESIPPVSFWRIMKLNLTEWPYFVVGVFC AIINGGLQPAFAVIFSKIIGIFTRNDDAETKRQNSNLFSLLFLVLGIVSFITFFLQGFTFGKAGEILTKRLRYMVFRSML RQDVSWFDDPKNTTGALTTRLANDAAQVKGAIGSRLAIITQNIANLGTGIIISLIYGWQLTLLLLAIVPIIAIAGVVEMK MLSGQALKDKKELEGAGKIATEAIENFRTVVSLTQEQKFEHMYDQSLQVPYRNSLRKAHIFGITFSFTQAMMYFSYAGCF RFGAYLVAHSLMSFEDVLLVFSAVVFGAMAVGQVSSFAPDYAKAKVSAAHIIMIIEKTPLIDSYSTEGLKPNTLEGNVTF NEVVFNYPTRLDIPVLQGLSLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPLAGKVLLDGKEIKQLNVQWLRAHLGIVS QEPILFDCSISENIAYGDNSRVVSQEEIVRAAKEANIHAFIESLPNKYSTRVGDKGTQLSGGQKQRIAIARALVRQPHIL LLDEATSALDTESEKVVQEALDKAREGRTCIVIAHRLSTIQNADLIVVFQNGRVKEHGTHQQLLAQKGIYFSMVSVQAGA KRQ (SEQ ID NO:334) etc.

[0193] The anti-MDR1 antibodies of the present invention exhibit high affinity binding to MDR1. For example, the anti-MDR1 antibodies of the present invention , at least about 10 -7 M, at least about 10 -8M, at least about 10 -9 M, or at least about 1 0 -10 The anti-MDR1 antibodies of the present invention bind to human MDR1 with an affinity of M. There are about 10 -7 M ~ about 10 -8 M, about 10 -8 M ~ about 10 -9 M, or about 10 -9 M ~ about 10 -10 Affinity of M Combine with.

[0194] The anti-MDR1 antibodies of the present invention may also bind to other proteins that are related but have different sequences, e.g. It does not substantially bind to epitopes formed by amino acids in different sequences of the EP. The anti-MD antibody targets an epitope formed by amino acids in proteins that have the same sequence but different amino acids. The binding of the anti-MDR1 antibody is generally substantially greater than the specific binding of the anti-MDR1 antibody to an epitope on MDR1. Substantially lower affinity generally refers to non-specific binding of at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower affinity.

[0195] The anti-MDR1 antibodies of the present invention inhibit the transport of molecules via MDR1 transporters (e.g., human MDR1). For example, the anti-MDR1 antibodies of the present invention can reduce the Compared to the degree of transport, the transport can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% %, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more can be further reduced.

[0196] In some embodiments, the subject antibodies include, for example, rodent, non-human primate, and human sequences. Therefore, the heavy chain FR coding sequence contains a FR region that is a mammalian sequence (e.g., encoded by the respective heavy chain FR coding sequence). (The one that was done).

[0197] This antibody showed 85% and 86% affinity to the sequences of the VH regions of the VH-VL pairs of the antibodies listed in Table 2. , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including 100%) The antibody may comprise a heavy chain variable (VH) region comprising an amino acid sequence identical to that of the antibody of interest (including the antibody of interest). The results were 85%, 86%, 87%, and 89% for the VL sequences of the VH-VL region pairs of the antibodies listed in Table 2. 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including 100%) The antibody may comprise a light chain variable (VL) region comprising an amino acid sequence that is identical to the antibody.

[0198] The regions and / or chains of the subject antibodies may be linked by one or more linker regions, If present, the linker region may be from about 5 amino acids to about 5 0 amino acids, for example, from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 It can be from about 45 aa to about 45 aa, or from about 45 aa to about 50 aa in length.

[0199] Linkers suitable for use in the subject antibodies include "flexible linkers." When used in conjunction with a linker molecule, the linker molecule generally has a size that allows some degree of flexible movement between the linked regions. The linker molecule is generally about 6 to 50 atoms long. For example, arylacetylene, ethylene glycol oligomers containing 2 to 10 monomer units. The diamines may be mers, diamines, diacids, amino acids, or combinations thereof. In light of the present disclosure, other linker molecules that can be attached to polypeptides may be used. It can be done.

[0200] Suitable linkers can be readily selected and may be of any suitable length. For example, it may be from 1 amino acid (e.g., Gly) to 20 amino acids, From 15 amino acids, from 3 amino acids to 12 amino acids, e.g., from 4 amino acids From 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids , or 7 to 8 amino acids, 1, 2, 3, 4, 5, 6, or may be 7 amino acids.

[0201] 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: 335) and GGGS n (SEQ ID NO: 336), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine-serine polymers are relatively unstructured, but both of these amino acids are This is of interest because it can act as a neutral tether between components. The amino acids have significantly more access to the phi-psi space than even alanine, and have longer side chains. Glycine polymers are of particular interest because they are far less constrained than residues (Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include Although not specified, GGSG (SEQ ID NO: 337), GGSGG (SEQ ID NO: 338), GSGSG (SE Q ID NO: 339), GSGGG (SEQ ID NO: 340), GGGSG (SEQ ID NO: 341), GSSSG (SEQ ID NO: 342) and the like. Those skilled in the art will appreciate that the design of peptides conjugated to any of the above elements is well within the skill of the art. , may comprise a linker that is wholly or partially flexible, such that the linker Inclusion of a flexible linker and one or more moieties that confer a less flexible structure You will realize that you can.

[0202] In another example, amino acid C220 can be mutated to serine or any other naturally occurring amino acid. Thus, by removing C220, by removing the entire hinge, or by removing Ig By replacing the G1 hinge with an IgG3 hinge, the flexibility of the hinge region of the antibodies of the present disclosure is increased. Antibodies are formed in which light chains are linked via their C-terminal cysteines, and This is similar to the situation seen with the human isotype IgA2m. This results in a higher Fc than Fc. This reduces the flexibility of the IgG1 molecule, resulting in a reduced cross-linking ability. The strategy is to replace the IgG1 hinge with an IgG2 hinge or an IgG2-like hinge. can introduce a variant of the IgG1 hinge that is similar to the IgG2 hinge. (TH7Δ6-9) contains a T223C mutation and two deletions (K222 and T225), resulting in an additional cysteine. This creates a short hinge.

[0203] Replacing the mouse CDRs into a human variable domain framework allows for their correct This can result in the retention of spatial orientation, for example, where the human variable domain framework is The CDRs have the same or similar conformation as the mouse variable domain framework from which they are derived. This approach is highly compatible with the mouse variable framework domains from which the CDRs are derived. Obtaining human variable domains from human antibodies with framework sequences that exhibit high degrees of sequence identity This can be achieved by using the same heavy and light chain variable framework regions. or different human antibody sequences. The human antibody sequences may be derived from the sequences of naturally occurring human antibodies. or may be a consensus sequence of several human antibodies. borough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering See neering 6:971 (1993).

[0204] Complementarity-determining regions of mouse donor immunoglobulins and appropriate human acceptor immunoglobulins Once a bridging antibody has been identified, the next step is to characterize it in order to optimize the properties of the resulting humanized antibody. Determining if and which residues should be replaced from these components Generally, the introduction of mouse residues prevents the antibody from becoming a human anti-mouse antibody (HAMA) in humans. Substituting human amino acid residues with mouse residues increases the risk of inducing a reaction. Art-recognized methods for measuring immune responses are can be performed to monitor HAMA responses in specific patients or during clinical trials. Patients receiving humanized antibodies will undergo immunogenicity assessment at the start of treatment and throughout the course of treatment. The HAMA response can be measured, for example, by surface plasmon resonance techniques (BIACORE) and / or or human IgG in serum samples from patients using methods known to those skilled in the art, including solid-phase ELISA assays. In many embodiments, the antibody response is measured by detecting antibodies against the therapeutic reagent. The subject humanized antibodies do not substantially induce a HAMA response in human subjects.

[0205] Specific amino acids from the human variable region framework residues contribute to the conformation of the CDRs. and / or selected for substitutions based on their possible effect on binding to the antigen. The unnatural juxtaposition of the mouse CDR regions and the human variable framework regions is a conformational This can result in restriction of the nucleotide sequence, unless corrected by substitution of specific amino acid residues. This leads to a loss of binding affinity.

[0206] The selection of amino acid residues for substitution is determined in part by computer modeling. Computer hardware for generating three-dimensional images of immunoglobulin molecules and software known in the art. Generally, molecular models are used to model immunoglobulins. It is constructed starting from the solved structure of a chain or its domains. The amino acid sequence similarity of the chains is compared with that of chains or domains of known three-dimensional structures, and the sequence is The chain or domain with the greatest similarity is selected as the starting point for building the molecular model. Chains or domains that share 50% sequence identity with each other are selected for modeling and are preferred. or those sharing at least 60%, 70%, 80%, 90% or more sequence identity are models. The solved starting structure is selected for the immunoglobulin chain or chains to be modeled. or to allow for differences between the actual amino acids in the domain and the amino acids in the starting structure. The modified structures are assembled into composite immunoglobulins. Finally, the energy By minimizing the energy and ensuring that all atoms are at the right distance from each other and that the bond lengths are Refine the model by verifying that the length and angle are within the chemically acceptable range. do.

[0207] In certain aspects, the subject antibodies comprise scFv multimers. For example, in some embodiments, In this case, the subject antibody may be an scFv dimer (e.g., containing two tandem scFvs (scFv2)), scFv trimers (e.g., containing three tandem scFvs (scFv3)), scFv tetramers (e.g., containing four tandem scFvs) tandem scFv (scFv4), or a multiplicity of more than four scFvs (e.g., in tandem) The scFv monomer is about 2 amino acids to about 15 amino acids in length, for example, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, also can be linked in tandem via a 15 aa linker. Suitable linkers include, for example, (Gly) x where x is an integer from 2 to 15. Other suitable linkers are those described above. In some embodiments, each of the scFv monomers in the subject scFv multimers is a human scFv monomer, as described above. In certain embodiments, the bispecific antibody is a hybrid of any molecular fragment known in the literature. A comprehensive overview of different bispecific antibody formats and production methods is provided. For a summary, see e.g. Brinkmann and Kontermann, MAbs. 2017, 9(2):182-212. 2017, 9 (2):182-212. For example, the bispecific antibodies of the present disclosure may be prepared using the methods described in Spiess C. et al., Mol Immunol. 2015 Oct; 67(2 Pt A):95-106. do.

[0208] In certain embodiments, the subject antibody comprises an immunoglobulin constant region (eg, an Fc region). The Fc region, if present, can be a human Fc region. If a constant region is present, the antibody may be a human The heavy chain constant region may include both the C1, hinge, C2, and C3 regions. The antibodies described herein include IgM, IgG, IgD, IgA, and IgM. All types of constant regions, including E, and all types of constant regions, including IgG1, IgG2, IgG3, and IgG4 An example of a suitable heavy chain Fc region is an antibody having the human isotype IgG1 F The light chain constant region can be lambda or kappa. A hybridized antibody may contain sequences from multiple classes or isotypes. As a tetramer containing a light chain and two heavy chains, as separate heavy chains, as light chains, Fab, Fa b'F(ab')2, and Fv, or heavy and light chain variable domains separated by a spacer. The antibody may be expressed as a single chain antibody linked by a single chain linker.

[0209] In some embodiments, the subject antibodies have a free thiol (-SH) at the carboxyl terminus. The free thiol group can be used to link the antibody to a second polypeptide (e.g., an antibody of the invention). The antibody may be used to bind to a target molecule (e.g., another antibody, including another antibody), scaffold, carrier, etc.

[0210] 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 polymer, Glutaraldehyde can be covalently attached to proteins (e.g., carbohydrates, toxins, etc.) via its amino moiety. The polypeptides are cross-linked using a homobifunctional cross-linking agent (e.g., a homobifunctional imide ester). ester, homobifunctional N-hydroxysuccinimidyl (NHS) ester, or homobifunctional Sulfhydryl-reactive crosslinkers (sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties and are used to crosslink the polymer. It can be used in a one-step reaction procedure in which the cross-linker is added to a solution containing a mixture of peptides. Homobifunctional NHS esters and imidoesters can be used to catalyze the synthesis of amine-containing polypeptides. At slightly alkaline pH, imidoesters react only with primary amines to form imidoesters. Homodimides are produced, and the overall charge of the crosslinked polypeptide is not affected. Functional sulfhydryl-reactive crosslinkers include bismaleimidohexane (BMH), 1,5-difluoro-2 ,4-Dinitrobenzene (DFDNB), and 1,4-Bis[3-(2-pyridyldithio)propionamide] Contains butane (DPDPB).

[0211] 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, such as Tris buffer, histidine buffer buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-methyl) 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt ( MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl solubilizers; detergents, such as Tween-20; may contain one or more of: non-ionic detergents such as; protease inhibitors; glycerol; can.

[0212] Compositions of the present disclosure also include pharmaceutical compositions comprising the antibodies described herein. An "effective amount" includes an effective amount of a subject antibody. It means a dose sufficient to reduce the rate of cancer growth, improve the symptoms of cancer, etc. The desired result is at least a reduction in cancer symptoms, a reduction in cancer growth, a decrease in the progression of cancer, or a decrease in the severity of cancer compared to a control. The subject antibodies can be delivered in a manner that avoids the blood-brain barrier, or may be formulated.

[0213] In some examples, the antibody may include a delivery enhancer, which - promotes passage of the blood-brain barrier, increasing permeability, e.g., allowing for efficient transdermal delivery This includes cases where progress can be made.

[0214] In some instances, the antibodies of the disclosure are not administered in a formulation with a delivery enhancer. In some instances, the antibodies of the present disclosure may themselves be capable of penetrating the blood-brain barrier. In some instances, the antibodies of the present disclosure may be used as anti-tumor agents, e.g., immunotherapeutics. as a delivery enhancer to facilitate penetration of the blood-brain barrier by anti-inflammatory or chemotherapeutic agents. In some examples, the antibodies of the present disclosure can be used in combination with, for example, another antibody or a chemotherapeutic agent. blood-brain barrier, blood-cerebrospinal fluid (CSF) barrier, blood-testis barrier or blood by active agents such as -Can be used as a delivery enhancer to facilitate crossing the placental barrier.

[0215] In this method, the antibody is any antibody capable of producing a desired therapeutic or diagnostic effect. The agent may be administered to the host using any convenient means. More particularly, the subject antibodies can be incorporated into a variety of formulations. It can be combined with a carrier or diluent to formulate into a pharmaceutical composition, and can be a solid, semi-solid, liquid, In solid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, It can be formulated into inhalant and aerosol preparations.

[0216] In pharmaceutical dosage forms, the subject antibodies may be administered together with a pharmaceutically acceptable excipient. or they, alone or in suitable association with other pharmaceutically active compounds, and combinations thereof. The following methods and excipients are merely exemplary. Yes, and it is by no means limiting.

[0217] The subject antibodies may be prepared from vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, etc. They can be dissolved, suspended or mixed in aqueous or non-aqueous solvents such as ethanol or propylene glycol. by emulsifying, and if desired by the addition of solubilizing agents, isotonicity adjusting agents, suspending agents, emulsifying agents, It can be formulated into an injectable preparation along with conventional additives such as stabilizers and preservatives. do.

[0218] Pharmaceutical compositions containing the subject antibodies can be prepared by dissolving the antibody with the desired purity in any physiologically acceptable medium. by mixing with carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents Acceptable carriers, excipients and / or stabilizers are used in the dosage form. and concentrations that are non-toxic to recipients and contain phosphate, citrate, and and other buffering agents such as organic acids; ascorbic acid, glutathione, cysteine, methionine and antioxidants, including citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenyl Alcohol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, chloride benzalkonium, or a combination thereof); arginine, glycine, ornithine, Lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, arachidonic acid phenylalanine, tyrosine, tryptophan, methionine, serine, proline and and their combinations; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (approx. (less than 10 residues) Polypeptides; proteins such as gelatin and serum albumin; sharpeners such as EDTA Additives: trehalose, sucrose, lactose, glucose, mannose, maltose , galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucose Sugars such as glucosamine, galactosamine, and neuraminic acid; and / or Tween, Bri j Nonionic surfactants such as Pluronic, Triton-X, and polyethylene glycol (PEG) include.

[0219] The pharmaceutical composition may be in liquid form, lyophilized form, or in liquid form reconstituted from a lyophilized form. The lyophilized formulation may be reconstituted with a sterile solution prior to administration.

[0220] Exemplary antibody concentrations in the pharmaceutical compositions are from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 100 mg / mL. It can be about 200 mg / mL, or in the range of about 150 mg / mL to about 200 mg / mL.

[0221] 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, about 5.0 to about 6.0, or about 5. The solution may be prepared at a pH in the range of 5. Examples of suitable buffer solutions for pH in this range include phosphate buffers. buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer and other organic acids The buffer concentration may vary depending, for example, on the buffer and the desired tonicity of the formulation. It can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0222] In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions are also suitable. The term "isotonic" refers to the relative concentration of a substance in a solution to which it is compared (e.g., physiological salt solution or The tonicity agent is a solution having the same tonicity as serum. The tonicity agent is added in an amount of about 5 mM to about 350 mM, for example, 10 It can be used in amounts of 0 mM to 350 nM.

[0223] reducing aggregation of the formulated antibody and / or minimizing particle formation in the formulation; and / or surfactants may also be added to the antibody formulation to reduce adsorption. Examples of the antibacterial agent include polyoxyethylene sorbitan fatty acid esters (Tween), ... Ethylene alkyl ether (Brij), alkylphenyl polyoxyethylene ether (Tr iton-X), polyoxyethylene-polyoxypropylene copolymer (poloxamer, Pluroxamer) Exemplary concentrations of surfactants include sodium dodecyl sulfate (SDS), and sodium dodecyl sulfate (SDS). The concentration can range from about 0.001% to about 1% w / v.

[0224] Protecting sensitive active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process For this purpose, a lyoprotectant may be added. Protective agents include sugars (including glucose and sucrose); polyols (mannitol, sorbitol and glycerol); and amino acids (alanine, glycine, glutathione, The lyoprotectant may be present in an amount of about 10 mM to 500 nM.

[0225] In some embodiments, a subject formulation comprises a subject antibody and one or more of the above-identified substances (e.g., For example, surfactants, buffers, stabilizers, and tonicity agents, and ethanol, benzyl alcohol phenol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben The present invention is essentially free from one or more preservatives, such as benzophenone, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is present in a concentration ranging from about 0.001 to about 2% (w / v), for example. Included in the formulation.

[0226] For example, the subject formulation may be a liquid or lyophilized formulation suitable for parenteral administration, containing about 1 mg / mL to about 200 mg / mL of the subject antibody; about 0.001% to about 1% of at least one detergent; about 1 mM about 100 mM buffer; optionally about 10 mM to about 500 mM stabilizer; and about 5 mM to about 305 mM tonicity. The solution may contain a surfactant and have a pH of about 4.0 to about 7.0.

[0227] The antibodies of the present invention can be utilized in aerosol formulations to be administered by inhalation. The antibody can be sprayed in pressurized acceptable propellants, such as dichlorodifluoromethane, propane, or nitrogen. It can be formulated as follows.

[0228] As used herein, the term "unit dosage form" refers to a single-dose formulation for human and animal subjects. "amount" refers to physically discrete units suitable for a given amount, each unit containing a pharmaceutically acceptable diluent, carrier, or the like. or a predetermined amount of the compound of the present invention calculated in an amount sufficient to produce the desired effect, together with a vehicle. The specifications for the subject antibody depend on the particular antibody used, the effect to be achieved, and the host The amount of antibody administered may depend on the pharmacokinetics associated with each antibody.

[0229] The subject antibodies can be administered as an injectable formulation. Typically, an injectable composition is a liquid. prepared as a liquid solution or suspension; dissolved or suspended in a liquid vehicle prior to injection Solid forms suitable for administration can also be prepared. The preparations may also be emulsified or The antibody may also be encapsulated in a liposome carrier.

[0230] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Additionally, if desired, 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 preparing dosage forms will be known, or apparent, to those skilled in the art.

[0231] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are In addition, pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, humectants, etc. Pharmaceutically acceptable adjuvants, such as lubricants, are also readily available to the public.

[0232] In some embodiments, the subject antibodies are formulated in a controlled release formulation. , can be prepared using methods well known in the art.

[0233] [Dosage and administration] The appropriate dosage should be determined by your doctor or other qualified healthcare professional based on various clinical factors. As is well known in the medical field, the dosage for a single patient can be determined by the individual. The amount depends on the patient's size, body surface area, age, the particular compound being administered, the patient's sex, time and Many factors, including the dosage and route of administration, general health, and other medications being administered at the same time, may affect your overall well-being. The antibody of interest may be administered at a dose ranging from 1 ng / kg body weight to 20 mg / kg body weight per dose, e.g., 0.1 mg / kg body weight to 1 0 mg / kg body weight, for example, between 0.5 mg / kg body weight and 5 mg / kg body weight; however, Doses below or above this exemplary range are also contemplated, particularly considering the factors mentioned above. If the dose is a continuous infusion, it may range from 1 μg to 10 mg / kg body weight / minute.

[0234] Those skilled in the art will appreciate that dose 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 dosage for a given compound may vary as a function of its receptivity. Dosages are readily determinable by those of skill in the art by a variety of means.

[0235] Distribution and excretion of monoclonal antibodies, in addition to dosing schedules based on body size Considering that the effect of body size is relatively small for See, e.g., Hendrikx et al, Oncologist 2017, 22(10):1212-1221 stomach.

[0236] [Administration route] The subject antibodies are administered to an individual using any available method and route suitable for drug delivery. This includes, for example, in vivo and ex vivo methods, as well as systemic and local routes of administration. Includes:

[0237] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, Subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes The routes of administration may be combined if desired, or the antibody and The subject antibody compositions may be administered in a single dose or multiple doses depending on the dosage and / or desired effect. In some embodiments, a subject antibody composition is administered orally. In some embodiments, a subject antibody composition is administered via the inhaled route. In some embodiments, a subject antibody composition is administered topically. In some embodiments, a subject antibody composition is administered intracranially. The substance is administered intravenously.

[0238] The drug may be administered via any available route suitable for delivery of conventional drugs, including systemic or local routes. Conventional methods and routes may be used to administer to a host. Routes of administration include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0239] Parenteral routes of administration other than inhalation include, but are not necessarily limited to, topical, transdermal, , subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., gastrointestinal Parenteral administration includes any route of administration other than via the oral route. Parenteral administration is the systemic or local administration of the subject antibody. When systemic delivery is desired, administration is typically performed invasively. This includes topical or mucosal administration of pharmaceutical preparations that are either soluble or systemically absorbed.

[0240] The subject antibodies can also be delivered to a subject by enteral administration. Enteral administration routes do not necessarily include: This includes, but is not limited to, oral and rectal (eg, using a suppository) delivery.

[0241] The subject antibodies are typically administered intravenously via parenteral (intravenous, subcutaneous, intramuscular, or intradermal) injection. The drug is administered by intravenous infusion or injection, preferably by subcutaneous injection. In patients receiving treatment for extended periods, subcutaneous administration offers significant benefits. Such benefits include patient convenience, short delivery time, and no need for dedicated injection equipment. Subcutaneous formulation and delivery is therefore advantageous for patient convenience and It can improve quality of life and provide cost savings to the healthcare system. For example, a subcutaneous fixed-dose combination containing two or more active molecules co-formulated in the same formulation. Alternatively, a multi-chamber device may be used in combination therapy. Inject multiple products sequentially or premix them directly before subcutaneous injection For further details, see, for example, Bittner et al, BioDrugs 2018, 32:425 -440; and Viola et al, J Control Release 2018, 286:301-314.

[0242] Treatment means at least an amelioration of symptoms associated with a pathological condition afflicting the host; Here, improvement refers to the improvement of the disease being treated, such as cancer and / or cancer growth and associated pain. To represent at least a decrease in the magnitude of a parameter (e.g., a symptom) related to a physical condition As such, treatment refers to the process by which the host is no longer in a pathological state or is at a reduced risk of developing a disease. The pathological condition or at least its associated symptoms must be eliminated so that the individual does not suffer from them. The associated symptoms are completely suppressed, e.g., prevented or stopped from occurring, e.g., This includes situations where the

[0243] Various subjects (wherein the term "subject" is used herein in conjunction with the terms "individual" and "patient") (used interchangeably with "anti-cancer drug" and "anti-cancer drug") are treatable according to the methods disclosed herein. Such subjects are "mammals" or "mammals," where these terms refer to animals that are meat-eaters. Order Voracious (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, rats), and Spirits Describes organisms belonging to the class Mammalia, including protozoa (e.g., humans, chimpanzees, and monkeys) In some embodiments, the host is a human.

[0244] Kits are provided having unit doses (e.g., oral or injectable doses) of the subject antibodies. In some embodiments, in addition to the container containing the unit dose, a There is an informational package insert describing the use of the antibody in treating rheumatoid arthritis and the associated benefits.

[0245] [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 in cells genetically engineered to secrete the nucleotide sequence It is linked to one or more regulatory elements, such as promoters and enhancers, that allow expression. They may be movably connected.

[0246] Suitable promoter and enhancer elements are known in the art. Suitable promoters for expression in bacterial cells include lacI, lacZ, T3, T7, gp These include, but are not limited to, t, lambda P, and trc. For this purpose, suitable promoters include, but are not limited to, light chain and / or is the promoter and enhancer element of the heavy chain immunoglobulin gene; cytomegalovirus Herpes simplex virus thymidine kinase promoter; Early and late SV40 promoters; promoters present in retroviral long terminal repeats. motor; mouse metallothionein-I promoter; and species known in the art Examples of such promoters include various tissue-specific promoters.

[0247] Nucleotide sequences encoding the subject antibodies are contained in expression vectors and / or cloning vectors. If the antibody of interest comprises two or more separate polypeptides, the two may be present in a vector. The nucleotide sequences encoding the polypeptides may be cloned into the same or separate vectors. Distinct polypeptides can be synthesized from a single nucleic acid or They can be expressed from a single vector, and strategies include using separate promoters, one or more They contain multiple internal ribosome entry sites (IRES), one or more self-cleaving sequences (e.g., 2A cleavage sites), Expression vectors include fragments of the ribosomal RNA (e.g., P2A, T2A, E2A, and F2A), and combinations thereof. The vector may contain selectable markers, origins of replication, and other components that provide for replication and / or maintenance of the vector. The features of the present invention may be included.

[0248] Large numbers of suitable vectors and promoters are known to those of skill in the art; many of which are of interest. The following vectors are provided as examples: Bacteria: pbs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, p NH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3 , pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pO G44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0249] Expression vectors generally provide for the insertion of nucleic acid sequences encoding heterologous proteins. It has convenient restriction sites located near the promoter sequence for the purpose of A selectable marker for use may be present. Suitable expression vectors include, but are not limited to: However, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus) ;Adeno-associated virus;SV40;Herpes simplex virus;Human immunodeficiency virus;Retroviral viral vectors (e.g., murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus) Viruses, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, bone marrow vectors derived from retroviruses such as proliferating sarcoma virus and mammary tumor virus) ;etc.

[0250] Nucleic acids, e.g., as described herein, may, in some instances, be used to transform, e.g., a cell into a The nucleic acid can be introduced into a cell by contacting the cell with the nucleic acid. These are generally referred to herein as genetically modified cells. These include, for example, naked nucleic acid delivery, viral delivery, chemical transfection, These include, but are not limited to, gene gun methods, gene gun techniques, and the like.

[0251] [cell] The present disclosure provides isolated, genetically modified cells (e.g., in vitro) that are genetically modified with a subject nucleic acid. In some embodiments, the isolated genetic composition is provided. The recombinant cells are capable of producing the antibody of interest. In some cases, the genetically modified cells are capable of producing the antibody of interest, e.g. The antibody can be delivered to a subject in need thereof. can be used in the production, screening, and / or discovery of multispecific antibodies.

[0252] Suitable cells include eukaryotic cells such as mammalian cells, insect cells, yeast cells, and bacterial cells. Introduction of a nucleic acid of interest into a host cell can be accomplished, for example, by the use of calcium phosphate buffer. precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection This can be done by injection, electroporation, or other known methods.

[0253] Suitable mammalian cells include primary cells and immortalized cell lines. 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, and 3T3 cells. , Vero cells, Huh-7 cells, BHK cells, PC12 cells, COS cells, COS-7 cells, RAT1 cells, mouse L cells, human embryonic kidney (HEK) cells, HLHepG2 cells, etc. .

[0254] In some instances, useful mammalian cells are derived from mammalian tissues or organs. In some instances, the cells used may include, for example, HEK 293T cells. Kidney cells, including kidney cells from established kidney cell lines such as:

[0255] In some examples, the cells of the present disclosure can be immune cells. The term "cells" generally refers to white blood cells derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, and "T cells" include helper T cells (CD4+ cells), cytotoxic T cells, and dendritic cells. CD3+ T cells, including cytotoxic T cells (CD8+ cells), regulatory T cells (Tregs), and gamma-delta T cells "Cytotoxic cells" include all types of immune cells that express cytotoxicity. These include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can

[0256] In some instances, useful cells expressing antibodies such as the multispecific antibodies of the present disclosure include: The antibodies may be engineered to contain nucleic acid sequences encoding the antibodies of the present disclosure. The generated productive T cells, in some instances, deliver the antibody to a subject in need thereof. It can be used for

[0257] In some examples, the immune cells of the present disclosure comprise an MDR1 binding domain, a transmembrane domain, and and immune effectors, including chimeric antigen receptors (CARs) containing intracellular signaling domains. -cells, wherein the MDR1-binding domain is a pair of variable heavy chains (VH) of an antibody listed in Table 2. H) heavy chain complementarity determining regions (HCDRs) and light chain CDRs (LCDRs) of the variable light chain (VL) region In one embodiment, the intracellular signaling domain comprises at least one co-stimulatory molecule. one or more functions derived from the gene (e.g., 4-1BB (i.e., CD137), CD27, and / or CD28) The intracellular signaling domain may comprise a functional signaling domain derived from a costimulatory molecule. Functional signaling domains derived from stimulatory molecules and functional signaling domains derived from stimulatory molecules It may include

[0258] The immune effector cells can be T cells. s) may be cells.

[0259] [method] As summarized above, the methods of the present disclosure include contacting cells with the antibodies of the present disclosure; A method of treating a subject according to the methods described herein, comprising administering to the subject the disclosed antibodies. elements (e.g., antibodies, multispecific antibodies, bispecific antibody molecules, compositions and formulations, nucleic acids, expression The present invention includes methods for producing the recombinant vectors, cells, etc.

[0260] As summarized above, the disclosed methods can detect the presence of MDR1 expression on, for example, cancer cells. to measure the level of MDR1 expression on cancer cells, or to promote and kill cancer cells. and / or enhance the immune response of cancer cells by contacting the cancer cells with an antibody of the present disclosure. In the example of , the killing of cancer cells is due to an immune response or immune cells acting on the cancer cells bound to the antibody. In some instances, cancer cell killing is mediated by MDR1 inhibition, e.g., by antibodies. The resulting inhibition of cellular egress of cancer cells is mediated by the Cell killing is achieved by inhibiting the cellular elimination of cancer cells plus immune-mediated responses (e.g., via the Fc region of antibodies). In some cases, cells contacted with a multispecific antibody The cancer cells may be multidrug resistant cancer cells. The method comprising contacting the cancer cells with the antibody of the present disclosure comprises: For example, the cancer cells may be treated with an additional therapy or active agent, including chemotherapy, immunotherapy, radiation therapy, etc. The method may or may not include contacting the

[0261] Contacting cancer cells with the multispecific antibodies of the present disclosure generally enhances the killing of cancer cells. (e.g., compared to the level of cancer cell killing in the absence of the multispecific antibody). In the case where an additional active agent is used, the results are compared with those observed using the additional active agent alone. Enhanced killing of cancer cells can be seen compared to the level of killing achieved by multispecific antibodies. The amount of enhancement in cancer cell killing that occurs varies, ranging from at least a 5% increase in cancer cell killing to a small increase in The increase may range from at least 90% to at least 90% or more, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least Such increases include, but are not limited to, increases of 80% or more. This may be compared to a single contact.

[0262] Enhanced killing of cancer cells has been demonstrated, for example, in observational studies, in vitro cell-based cytotoxicity assays, and in vitro cell-based cytotoxicity assays. cytometry, flow cytometry, cell viability labeling (e.g., using one or more cell viability stains) The present invention may be assessed by a variety of means, including but not limited to, the use of steroids (e.g., steroid hormones ...

[0263] [Treatment method] The present disclosure provides methods for treating cancer (particularly MDR1-expressing or MRD-overexpressing cancers), The method generally involves administering to an individual in need thereof (e.g., an individual with such cancer) An effective amount of an antibody provided herein may be administered alone (e.g., in monotherapy) or in combination with one or more additional This includes administering the antibody of the present disclosure in combination with an additional therapeutic agent (e.g., in combination therapy). Administration can be by any convenient and suitable delivery route.

[0264] Thus, administering includes, for example, delivery of an antibody by injection, delivery of an antibody by infusion, etc. delivery of nucleic acids or expression vectors encoding the antibodies; targeting cells that express and secrete the antibodies. Delivery of antibodies by administration to the cytoplasm, MDR1-binding domain, transmembrane domain, and intracellular signaling Immune effectors expressing chimeric antigen receptors (CARs) containing transduction domains on the cell surface Delivery of cells (e.g., CAR-T cells) (wherein the MDR1-binding domain is an antibody listed in Table 2) These include, but are not limited to, HCDRs and LCDRs of the VH and VL domain pairs. Administration of a drug, a nucleic acid encoding a drug, a cell expressing a drug, etc., involves contacting the drug with the drug. This may include contacting with a nucleic acid, contacting with a cell, etc.

[0265] In some embodiments, an effective amount of a subject antibody is administered alone ( in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy) ) compared to the severity of adverse symptoms in the absence of treatment with the antibody. at least about 5%, at least about 10%, at least about 15%, at least about 20%, At least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about Reduce by 60%, at least about 70%, at least about 80%, at least about 90%, or more It is an effective amount.

[0266] In some embodiments, an effective amount of a subject antibody is administered alone (e.g., as a single agent) in one or more doses. therapy) or in combination with one or more additional therapeutic agents (e.g., combination therapy) In some cases, ameliorating the cancer in the treated individual (e.g., slowing the growth of the cancer, Stopping the growth of cancer cells, reversing the growth of cancer, and killing cancer cells (including tumor cells) For example, an effective amount of a subject antibody is an amount effective for the treatment of a subject in the absence of treatment with the antibody. at least about 5%, at least about 10%, at least about 15%, at least about 20%, At least about 25%, at least about 30%, at least about 40%, at least about 50%, or more and may slow the rate of cancer growth or reduce the size of the cancer in an individual. do.

[0267] In some instances, the subject is administered a subject with or without one or more additional reagents. The present invention relates to a systemic treatment, including the use of antibodies against the inflammatory cytokines. " refers to a specific tumor (e.g., a primary tumor or a defined secondary tumor) or a specific cancer. Targeting only the containing tissue (e.g., liver for liver cancer, blood for blood cancer, etc.) Systemic treatment generally refers to treatment that is directed at the entire body of a subject, e.g. For example, this may include systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof, etc. However, it is not limited to these.

[0268] In some instances, the subject is administered with or without one or more additional reagents: The subject may be treated locally, including with a subject antibody. "Location" refers to a tumor that is specifically directed to the location of the tumor (e.g., a primary tumor or a defined secondary tumor). treatment or cancer-containing tissue (e.g., liver for liver cancer, blood for blood cancer) In some instances, localized treatment also refers to treatment specifically directed at a target area (e.g., a stool ... Affects the environment surrounding the tumor (e.g., the tissues surrounding the tumor, e.g., the tissues immediately adjacent to the tumor) Localized treatments generally involve administering the drug to the tumor (e.g., the primary tumor). Tissues distant from the site of cancer, including the site of the cancer, are not affected or are not targeted there. It is a useful local treatment that can be administered in addition to or in combination with the subject antibodies. These include, for example, surgery, local radiation therapy, local cryotherapy, local laser therapy, and local topical therapy. These include, but are not limited to, methods, combinations thereof, etc.

[0269] In certain embodiments, a subject treatment method comprises administering a subject antibody and one or more additional therapeutic agents. Suitable additional therapeutic agents include chemotherapeutic agents, radiotherapy agents, immunotherapy agents, and the like. These include, but are not limited to, reagents, other antibodies, or multispecific antibody agents. Additional treatments that may be administered to the subject before, during, or after administering the multispecific antibodies of the present invention include: , including, for example, the type of cancer, the subject's medical history, general health status and / or comorbidities, etc. It depends on many factors. Useful cancer treatments include, for example, radiation therapy, chemotherapy, immunotherapy, and the like. These include, but are not limited to, therapy.

[0270] Radiation therapy can be delivered from an externally applied source such as, but not limited to, a beam. or x-rays or gamma rays delivered by implantation of a small radioactive source.

[0271] Antibodies suitable for use in cancer therapy include naked antibodies, e.g., trastuzumab (Hermann's bevacizumab (Avastin™), cetuximab (Erbitux™), Nitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Ri tuxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), oregovomab (OvaRex™), lambrolizumab (MK-3475), pertuzumab (Perjet a™), ranibizumab (Lucentis™) and conjugated antibodies, e.g. Gemtuzumab ozogamicin (Mylortarg™), brentuximab vedotin (Adcet ris™), 90Y-labeled ibritumomab tiuxetan ((Zevalin™), 131I-labeled These include, but are not limited to, Tositumoma (Bexxar™), and the like.

[0272] 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, CD52, EpCA, M, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding protein, ganglioside ( (e.g., GD2, GD3, GM2, etc.), Ley, VEGF, VEGFR, integrin alpha-V-beta-3, Integrin alpha-5-beta-1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, T RAILR2, RANKL, FAP, tenascin, programmed cell death 1 ligand 1 (PD-L1), androgen Antibody receptor (AR), Bruton's tyrosine kinase (BTK), BCR-Abl, c-kit, PIK3CA, EML4-ALK , KRAS, ALK, ROS1, AKT1, BRAF, MEKJ, MEK2, NRAS, RAC1, ESR1, CTLA-4, LAG-3 and These antibodies include, but are not limited to, TIM-3 and the like. as a combination therapy with anti-MDR1 antibodies, or with at least one of these antibodies and the antigen-binding portion of the anti-MDR1 antibody provided herein. can be given.

[0273] Conventional cancer therapies also include targeted therapies for cancer, such as: These include, but are not limited to: Ado-trastuzumab, which targets HER 2 (ERBB2 / neu) Emtansine (Kadcyla) (approved for use in breast cancer); EGFR (HER1 / ERBB1), H Afatinib (Gilotrif), which targets ER2 (ERBB2 / neu), is approved for use in non-small cell lung cancer. targeted aldesleukin (Proleukin) (for renal cell carcinoma, melanoma) approved for use; alectinib (Alecensa), which targets ALK (for non-small cell lung cancer) approved for use in chronic B-cell lymphoma; alemtuzumab (Campus) targeting CD52 ( approved for use in lymphocytic leukemia; atezolizumab (Tesen), which targets PD-L1 Triclosan (approved for urothelial cancer and non-small cell lung cancer); avelumab (Bavencio) (licensed for use in Merkel cell carcinoma); KIT, P Axitinib (Inlyta), which targets DGFRβ and VEGFR1 / 2 / 3 (approved for use in renal cell carcinoma) belimumab (Benlysta), which targets BAFF (used in lupus erythematosus); approved); belinostat (Beleodaq) which targets HDAC (peripheral T-cell lymphoma) bevacizumab (Avasti), which targets VEGF ligands; (cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma, non-small cell lung cancer; ovarian cancer, peritoneal cancer, renal cancer) approved for use in cancers of the ovarian and ovarian cancers; blinatumomab (Bili) targeting CD19 / CD3 Incyte) (approved for use in acute lymphoblastic leukemia (precursor B cell); Bortezomib (Velcade), which targets the proteasome (multiple myeloma, mantle cell approved for use in lymphoma; bosutinib (Bosulif), which targets ABL (approved for use in chronic myeloid leukemia); brentuximab, which targets CD30 Buvedotin (Adcetris) (use in Hodgkin's lymphoma, anaplastic large cell lymphoma) has been approved; brigutinib (Alumbrig) targeting ALK (non-small cell lung cancer (AL) approved for use in patients with glaucoma (including those with glaucoma, psoriasis, and rheumatoid arthritis); Caboza, which targets FLT3, KIT, MET, RET, and VEGFR2 entinib (Cabometyx, Cometriq) (approved for use in medullary thyroid cancer and renal cell carcinoma) carfilzomib (Kyprolis), which targets the proteasome (multiple bone marrow disease) approved for use in myeloma; ceritinib (Zykadia), which targets ALK (non- approved for use in small cell lung cancer; cetaxel targeting EGFR (HER1 / ERBB1) Mab (Erbitux) (approved for use in colorectal cancer and squamous cell carcinoma of the head and neck) cobimetinib (Cotellic), which targets MEK (approved for use in melanoma) Crizotinib (Xalkori), which targets ALK, MET, and ROS1 (for non-small cell lung cancer) approved for use in the treatment of BRAF-targeted leukemia; dabrafenib (Tafinlar) (Metformin); approved for use in non-small cell lung cancer (NSCLC); CD38-targeted daratumoma Darzalex (approved for use in multiple myeloma); ABL-targeting Dasatinib (Sprycel) (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia) Denosumab (Xgeva), which targets RANKL (use in giant cell tumor of bone) is approved); dinutuximab (Unituxin) targeting B4GALNT1 (GD2) (pediatric approved for use in neuroblastoma; durvalumab (I) targeting PD-L1 Miffinzi) (approved for use in urothelial carcinoma); SLAMF7 (CS1 / CD319 / CRACC) elotuzumab (Empliciti), which targets enasidenib (Idhifa), which targets IDH2 (used in acute myeloid leukemia) approved); erlotinib (Tarceva) targeting EGFR (HER1 / ERBB1) (non-small cell lung cancer) approved for use in alveolar lung cancer and pancreatic cancer; everolimus (African-derived steroid hormone), which targets mTOR initor) (neuroendocrine tumors of pancreatic, gastrointestinal or pulmonary origin, renal cell carcinoma, unresectable subependymal tumors Approved for use in giant cell astrocytoma and breast cancer; targets EGFR (HER1 / ERBB1) Gefitinib (Iressa) (approved for use in non-small cell lung cancer); Targeted ibritumomab tiuxetan (Zevalin) in non-Hodgkin's lymphoma approved for use; ibrutinib (Imbruvica), which targets BTK (mantle cells) in follicular lymphoma, chronic lymphocytic leukemia, and Waldenström's hypergammaglobulinemia approved for use; idelalisib (Zydelig) targeting PI3Kδ (chronic lymphoma) Approved for use in leukemia, follicular B-cell non-Hodgkin's lymphoma, and small lymphocytic lymphoma Imatinib (Gleevec) targets KIT, PDGFR, and ABL (GI stromal tumors (KIT+) approved for use in dermatofibrosarcoma protuberans, dermatofibrosarcoma protuberans, and multiple hematologic malignancies; targeting CTLA-4 ipilimumab (Yervoy) (approved for use in melanoma); Ixazomib (Ninlaro), which targets the teasome, is approved for use in multiple myeloma lapatinib (Tyke) targets HER2 (ERBB2 / neu) and EGFR (HER1 / ERBB1). Lube (approved for use in breast cancer (HER2+)); Lenvatin, which targets VEGFR2 Lenvima (approved for use in renal cell carcinoma and thyroid cancer); Midostaurin (Rydapt), approved for use in acute myeloid leukemia (FLT3+) ); EGFR (HER1 / ERBB1)-targeting necitumumab (Portrazza) (non-small cell lung cancer) approved for use in lung cancer; neratinib (Ner lynx) (approved for use in breast cancer); nilotinib (Tasigna), which targets ABL ) (approved for use in chronic myeloid leukemia); niraparib, which targets PARP ( Zejula) (approved for use in ovarian cancer, fallopian tube cancer, and peritoneal cancer); targeting PD-1 Nivolumab (Opdivo) (colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, approved for use in hepatoma, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma); CD20 Obinutuzumab (Gazyva), which targets ofatumumab (Arzerra, HuMax-CD20), which targets CD20; (approved for use in chronic lymphocytic leukemia); olaparib, which targets PARP ( Lynparza (approved for use in ovarian cancer); Oralazur, which targets PDGFRα Lartruvo (approved for use in soft tissue sarcomas); an EGFR-targeted Simertinib (Tagrisso) (approved for use in non-small cell lung cancer); CDK4, CD palbociclib (Ibrance), which targets K6 (approved for use in breast cancer); Panitumumab (Vectibix) targets EGFR (HER1 / ERBB1) in colorectal cancer panobinostat (Farydak), which targets HDAC (many approved for use in multiple myeloma; pazopanib, which targets VEGFR, PDGFR, and KIT Votrient (approved for use in renal cell carcinoma); a peptide inhibitor targeting PD-1 Mubrolizumab (Keytruda) (classical Hodgkin lymphoma, melanoma, non-small cell lung cancer) (PD-L1+), head and neck squamous cell carcinoma, solid tumors (MSI-H); HE Pertuzumab (Perjeta), which targets R2 (ERBB2 / neu) and is used in breast cancer (HER2+) approved); ponatinib (Iclusig) targeting ABL, FGFR1-3, FLT3, and VEGFR2 ) (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); VEGFR2 Targeted ramucirumab (Cyramza) for colorectal cancer, gastric cancer, or esophagogastric junction (GEJ) cancer approved for use in cancer and non-small cell lung cancer); KIT, PDGFRβ, RAF, RET, VEGFR1 / 2 Regorafenib (Stivarga) targeting β-amyloid β-amyloid 3 (colorectal cancer, gastrointestinal stromal tumors, hepatocellular carcinoma) approved for use in: ribociclib (Kisqali), which targets CDK4 and CDK6; approved for use in breast cancer (HR+, HER2-); rituximab (Ritux) targeting CD20 Tuxan, MabThera) (non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, multiple myeloma) approved for use in vasculitic granulomatosis; rituximab / hibuprofen, which targets CD20; Alanylase, human (Rituxan Hycela) (chronic lymphocytic leukemia, diffuse large cell type B) approved for use in cytoplasmic lymphoma, follicular lymphoma; Depsin (Istodax) (for use in cutaneous T-cell lymphoma and peripheral T-cell lymphoma) approved; PARP-targeting rucaparib (Rubraca) (approved for use in ovarian cancer) Ruxolitinib (Jakafi), which targets JAK1 / 2 (used in myelofibrosis) approved for use in patients with IL-6-targeted IL-6-associated thrombocytopenia (multicentric capsid). Approved for use in spleen disease; targeted sipuleucel-T (Provenge) (previously approved for use in prostate cancer; smoothened targeting sonidegib (Odomzo) ) (approved for use in basal cell carcinoma); targets VEGFR, PDGFR, KIT, and RAF Sorafenib (Nexavar) (approved for use in hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer) temsirolimus (Torisel), which targets mTOR (approved for use in renal cell carcinoma) Tositumomab (Bexar), which targets CD20 (for non-Hodgkin's lymphoma) approved for use in the treatment of rheumatoid arthritis; trametinib (Mekinist), which targets MEK (Melanoma) approved for use in non-small cell lung cancer; targets HER2 (ERBB2 / neu) Trastuzumab (Herceptin) (approved for use in breast cancer (HER2+) and gastric cancer (HER2+) Vandetanib (Caprelsa) (A) targets EGFR (HER1 / ERBB1), RET, and VEGFR2. approved for use in medullary adenomatous carcinoma; vemurafenib (Zerbo) targeting BRAF raf) (approved for use in melanoma); venetoclax, which targets BCL2 (Veneclexta) (approved for use in chronic lymphocytic leukemia); PTCH, Sm Vismodegib (Elevage), which targets the vasodilator, is approved for use in basal cell carcinoma. vorinostat (Zolinza), which targets HDAC (in cutaneous T-cell lymphoma) approved for use; Ziv-aflibercept (Zaltra), which targets PIGF and VEGFA / B tbsp) (approved for use in colorectal cancer); etc. These antibodies are It may be administered as a combination therapy with the provided anti-MDR1 antibodies.

[0274] Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to: (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonist; (5) interleukin-2; (6) interferon (7) interferon-γ; (8) colony-stimulating factors; (9) angiogenesis inhibitors; and (10) Including tumor necrosis factor antagonists.

[0275] Chemotherapeutic or antineoplastic agents are non-peptide (i.e. Non-proteinaceous compounds, including cytotoxic and cytostatic agents. Non-limiting examples of agents include alkylating agents (e.g., nitrosoureas), antimetabolites (e.g., methotrexate), and the like. Rexate), antitumor antibiotics (e.g., anthracyclines), plant alkaloids (e.g., (e.g., vinca alkaloids, taxanes), topoisomerase inhibitors, and steroid hormones This includes Mont.

[0276] Agents that act to reduce cell proliferation are known in the art and are widely used. Such agents include nitrogen mustard, nitrosoureas, and ethylene Alkylating agents such as imine derivatives, alkyl sulfonates, and triazenes. , including but not limited to mechlorethamine, cyclophosphamide (Cytoxa lon (trademark), melphalan (L-sarcolysin), carmustine (BCNU), lomustine ( CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil Stard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenediamine thiaminol, triethylenethiophosphoramine, busulfan, dacarbazine, and temo Zolomid is an example.

[0277] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine analogs. amidocycline inhibitors, including but not limited to cytarabine (CYTOSAR-U) , cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thiazol-2-one Oguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU ), methotrexate, 10-propargyl-5,8-dideazaphorate (PDDF, CB3717), 5,8- Dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostaphylococcus aureus These include fluoxetine, and gemcitabine.

[0278] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes) , lymphokines, epipodophyllotoxins) such as Ara-C, paclitaxel (Taxon) Docetaxel (Taxotere®), deoxycodone (Taxotere®), Synthin, mitomycin-C, L-asparaginase, azathioprine; brequinar; e.g., bile Alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine; Antibiotics, such as anthracyclines; Daunorubicin hydrochloride (daunomycin, rubidomycin, cerbidine), idarubicin doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxyzinccyclopentasiloxane peptides, such as dactinomycin; basic glycopeptides such as bleomycin; anthracyclines Non-glycosides, e.g., plicamycin (mithramycin); anthracenediones, e.g., mithramycin Toxantrone; azirinopyrroloindole diones such as mitomycin; cyclosporin; macrocyclic immunosuppressants such as thiazolinone, FK-506 (tacrolimus, Prograf), and rapamycin; These include, but are not limited to:

[0279] Other antiproliferative cytotoxic agents include navelbene, CPT-11, anastrazole, and letrazole. , capecitabine, reloxafine, cyclophosphamide, ifosfamide, and dolomide It is Xafin.

[0280] Microtubule-interacting agents with antiproliferative activity are also suitable for use, including, but not limited to: However, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (N SC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), Itansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol ( Taxol® derivatives, docetaxel (Taxotere® )), thiocolchicine (NSC 361792), trityl cysterol, vinblastine sulfate, sulfate Vincristine, including but not limited to eoptilon A, epothilone B, and discodermolide Natural and synthetic epothilones, including but not limited to estramustine, nocodazole, etc. .

[0281] Suitable hormone regulators and steroids (including synthetic analogues) for use include adrenal Corticosteroids, such as prednisone, dexamethasone; estrogens and pregesins cin, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, vinegar megestrol, estradiol, clomiphene, tamoxifen, etc.; and adrenal glands Cortical depressants, such as aminoglutethimide; 17α-ethynylestradiol; diethylsulfonyl Chilbestrol, testosterone, fluoxymesterone, dromostanoate propionate Lon, testolactone, methylprednisolone, methyltestosterone, prednisolone , triamcinolone, chlorotrianisene, hydroxyprogesterone, aminogluteth amide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide ( Drugs such as fluticasone (Drogenil), toremifene (Fareston), and Zoladex Estrogen stimulates proliferation and differentiation, so estrogen receptors Compounds that bind to the body are used to block this activity. It can inhibit the proliferation of cells.

[0282] Other chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, etc. ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidote Phyllotoxins;Topoisomerase inhibitors;Procarbazine;Mitoxantrone;Leuco Other antiproliferative agents include immunosuppressants such as micofur, thiazolinone ... phenolic acid, thalidomide, deoxyspergualin, azasporin, leflunomide, Zolivine, azaspiran (SKF 105685), etc.; Iressa (registered trademark) (ZD 1839, 4-(3-chloro- (b)-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazolidinyl ; etc.

[0283] "Taxanes" include paclitaxel as well as other active taxane derivatives or prodrugs. "paclitaxel" (e.g., docetaxel, Taxol®, Taxol Sotaire™ (a formulation of docetaxel), a 10-desacetyl analog of paclitaxel and and analogues such as the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analogue of paclitaxel The term "antibody" should be understood herein to include compounds, formulations and derivatives thereof, known to those skilled in the art. and can be readily prepared using techniques such as those described in WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Patent Nos. 5,294,637; 5,283,253; 5,279, See also EP 590,267; EP 590,267; EP 590,267; EP 590,267; EP 590,267; EP 590,267; EP 590,534 ...534; EP 5 (see reference) or various distributors, such as Sigma Chemical Co., St. Louis, Missouri. It can be obtained from the company (T7402 from Taxus brevifolia; or Taxus yannanens is derived from T-1912).

[0284] Paclitaxel refers to the common chemically available forms of paclitaxel as well as Analogs and derivatives (e.g., the above-mentioned Taxotere™ docetaxel) and paclitaxel Cell conjugates (e.g., Abraxane (albumin-bound), paclitaxel-PEG, paclitaxel It is understood to also refer to paclitaxel-dextran, or paclitaxel-xylose). should be.

[0285] The term "taxane" also includes various publicly known compounds, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include those described in International Patent Application WO 99 / 18113. galactose and mannose derivatives described in WO 99 / 14209; piperazino derivatives described in WO 99 / 14209; and other derivatives; as described in WO 99 / 09021, WO 98 / 22451 and U.S. Pat. No. 5,869,680. taxane derivatives described in WO 98 / 28288; 6-thio derivatives described in U.S. Pat. No. 5,821,221; 63; and sulfenamide derivatives described in U.S. Pat. No. 5,415,869. These include, but are not limited to, sole derivatives. 59; and paclitaxel products, including those described in U.S. Pat. No. 5,824,701. These include, but are not limited to, rugs.

[0286] Useful immunotherapies include: anti-PD-1 / PD-L1 immunotherapy, and / or or other immunotherapy targets that can be targeted in a therapeutic method, such as CTLA-4, LAG-3, and and immune checkpoint markers such as TIM-3. Anti-PD-1 / PD-L1 immunotherapy includes, e.g. including, but not limited to, administering an effective amount of one or more therapeutic anti-PD-1 / PD-L1 antagonists. Therapies include those comprising administering to a subject, such antagonists as Opdivo. nivolumab, Keytruda (pembrolizumab), and Tecno Intrik™ (atezolizumab), durvalumab (MEDI4736), avelumab (MS B0010718 C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242, etc. These antibodies include, but are not limited to, the anti-MDR1 antibodies provided herein. It may be administered as a combination therapy with an antibody.

[0287] CTLA-4, also known as CD152, binds to CD80 and CD86. Antibodies against CTLA-4 are , which is approved for the treatment of some types of cancer. Due to its inhibitory effect, CTLA-4 can be used in combination with other immunotherapies to treat certain cancers. TIM-3 is also a good candidate for immunotherapy against several cancer types. can be targeted for

[0288] LAG-3 is in clinical trials for the treatment of cancer. Anti-LAG-3 immunotherapy involves down-regulating LAG-3. by inhibiting signals to pre-activated LAG-3+ cells. Activating Treg cells while suppressing induced (i.e., antigen-specific) Treg suppressive activity Useful LAG-3 antagonists include those that utilize antagonist LAG-3 antibodies that can inhibit the The target antibody was relatlimab (BMS-986016; developed by Bristol-Myers Squibb). ), IMP701 (developed by Immutep), TSR-033 (anti-LAG-3 mAb; developed by TESARO, Inc. (developed by

[0289] Immunotherapies include, for example, adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T-cell therapy. For example, the subject may be administered a T cell-based immunotherapy that targets a gene that is expressed by the subject's cancer. A population of CAR T cells engineered to target the antigen of interest can be administered. Therapy may, in some instances, involve obtaining a cell sample from the subject, such as a blood sample or tumor biopsy, and then Ex vivo cultivation of immune cells from a sample (including genetic modification of cultured immune cells) In one example, immune cells can be obtained from a subject and ex vivo The cells are cultured in a lab and engineered with a CAR specific to an antigen expressed by the cancer, resulting in CAR T cell recruitment. The CAR T cells can then be reintroduced into the subject to target the cancer. Cell-based immunotherapy can be performed in a variety of ways, depending on the particular cancer being treated, e.g., by using various antigens. This can be achieved by targeting different sources, harvesting / culturing different cell types, etc. Furthermore, T cell-based immunotherapies can be administered systemically (e.g., by intravenous injection) or locally. (e.g., injection (e.g., intraperitoneal injection, thoracic catheter injection, etc.), direct injection, etc.) can be administered.

[0290] In some instances, the therapeutic methods described herein involve targeting one or more of the multidrug resistance transporters. The method may include administering one or more inhibitors to a subject, for example, but not limited to, For example, multidrug resistance transporters other than MDR1 are included. Inhibitors include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and low-molecular-weight inhibitors. These include molecular inhibitors of multidrug resistance transporters, such as ABC transporters Inhibitors are included.

[0291] Individuals suitable for treatment using the methods of the present disclosure include individuals with cancer; individuals diagnosed with cancer; Individuals undergoing cancer treatment, such as chemotherapy, radiation therapy, antibody therapy, or surgery; have received chemotherapy, radiation therapy, antibody therapy, surgery, or other treatments individuals who have not responded to treatment; individuals who have not responded to cancer treatment (e.g., chemotherapy, radiation therapy, antibody therapy, surgery, etc.) have initially responded to treatment but subsequently relapsed, i.e., This includes individuals whose cancer has recurred.

[0292] The disclosed methods can be used to treat a variety of cancers, including, for example, primary cancers, secondary cancers, regrowing cancers, recurrent cancers, refractory cancers, and the like. For example, in some instances, the present invention can be used to target and treat various cancers. The methods described herein can be used as initial treatment for a primary cancer identified in a subject. In examples, the methods of the disclosure can be used as a non-primary (e.g., second-line or later) treatment, e.g., For example, in subjects with cancer that has not responded to prior treatment, the use of In subjects who have a mixed reaction to a previous treatment (e.g., at least one Positive response to the tumor and negative response to at least one second tumor in the subject It can be used in subjects with a negative or neutral response.

[0293] In some examples, the methods of the disclosure include treating a subject with a drug-resistant cancer, such as a multidrug-resistant cancer. Multidrug resistance (MDR) is a condition in which many cancers become resistant to chemotherapy drugs. This mechanism minimizes cell death and promotes the growth of drug-resistant tumors. These include, but are not limited to, increased expression of efflux pumps, decreased drug absorption, One or more resistance mechanisms may be involved, including inhibition of cell death or apoptosis, modulation of drug metabolism, etc. In some examples, the methods of the disclosure may prevent, reverse, or circumvent MDR. It is possible.

[0294] In some examples, the methods of the disclosure include effectively treating a subject with a cancer that is resistant to a first agent. and treating the patient with an amount of an antibody described herein in combination with a second agent that is different from the first agent. For example, in some instances, the subject's cancer is resistant to a first chemotherapy. and the subject administers an effective amount of an antibody described herein to a second chemotherapy agent different from the first. For example, the type of cancer being treated, resistance to Various combinations of first and second chemotherapy agents are used depending on the likelihood of developing cancer. It can be done.

[0295] Many cancers are known to develop drug resistance. For this and other reasons, Therefore, the disclosed methods can be used to treat a variety of cancers, including, but not limited to: It can find use in: Acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocorticoblastoma, AIDS-related cancers (e.g. (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendix cancer, astrocytoma, atypical teratoid rhabdominis Hepatic tumors, basal cell carcinoma, cholangiocarcinoma (extrahepatic), bladder cancer, bone cancer (e.g. Ewing's sarcoma, osteosarcoma and malignant fibrous histiocytoma, etc.), brainstem glioma, brain tumor (e.g., astrocytoma, central nervous system embryonal tumor) , central nervous system germ cell tumors, craniopharyngioma, ependymoma, etc.), breast cancer (e.g., female breast cancer, male breast cancer) cancer, pediatric breast cancer, etc.), bronchial tumors, Burkitt's lymphoma, carcinoid tumors (e.g., pediatric stage, gastrointestinal system, etc.), occult primary tumors, cardiac (heart) tumors, central nervous system (e.g. atypical malformations) rhabdoid tumor, embryonal tumor, germ cell tumor, lymphoma, etc.), cervical cancer, childhood cancer, notochordal cancer tumors, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid tumors, colon cancer, Intestinal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal (e.g. bile duct, extrahepatic, etc.), breast in situ Ductal carcinoma (DCIS), embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, head Extrahepatic 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 cancer, Gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial, gonadal) extrauterine, ovarian, testicular, etc.), gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer , hepatocellular (liver) carcinoma, histiocytosis (e.g., Langerhans cell, etc.), Hodgkin's lymphoma , hypopharyngeal cancer, intraocular melanoma, islet cell tumors (e.g., pancreatic neuroendocrine tumors, etc.), Kaposi's carcinoma tumors, kidney cancers (e.g., renal cell, Wilms tumor, childhood renal tumors, etc.), Langerhans cell histiocytosis leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CRL), and leukemia (LL). lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hair cell, etc.), lip / oral cancer, liver Cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), lymphoma ( e.g., AIDS-associated, Burkitt's, cutaneous T-cell, Hodgkin's, non-Hodgkin's, primary central nervous system ( CNS), etc.), macroglobulinemia (e.g., Waldenstrom's disease, etc.), male breast cancer, malignant Osteofibrous histiocytoma and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastases of unknown primary origin Metastatic squamous cell cervical cancer, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, Myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm , myeloid leukemia (e.g., chronic myeloid leukemia (CML), etc.), myeloid leukemia (e.g., acute myeloid leukemia (AML), etc.), bone marrow leukemia Myeloproliferative tumors (e.g., chronic, etc.), nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-homologous Lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer (e.g., lip cancer), oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma of the breast and bone, ovarian cancer (e.g., epithelial, germ cell tumors, low-grade tumors, etc.) ), pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomas, paragangliomas, sinus and nasal cancers , parathyroid carcinoma, 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 ureter, transitional cell carcinoma , retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcomas (e.g., Ewing, Kaposi, osteosarcoma, striated muscle sarcoma, soft tissue, uterine, etc.), Sézary syndrome, skin cancer (e.g., childhood, melanoma, Lung cancer, small intestine cancer, soft tissue cancer, squamous cell carcinoma, etc. tumors, squamous cell cervical cancer (e.g., occult primary, metastatic, etc.), gastric cancer, T-cell lymphoma, testicular cancer , throat cancer, thymoma and thymocytoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureter and renal pelvis cancer , urethral cancer, uterine cancer (e.g., endometrial, etc.), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström Hemoglobulinemia, Wilms' tumor, etc.

[0296] The treatment methods described herein may, in some instances, be in addition to one or more conventional treatments. For example, in the case of oncology, the methods described herein may be performed on subjects who have previously undergone In some instances, conventional cancer treatments (e.g., but not limited to, conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc. In some instances, the methods described herein may be used to treat a condition in which a subject has not responded to conventional treatment. In some instances, the methods described herein may be used when the patient has not received or is refractory to treatment. The described methods can be used when a subject has responded to conventional treatment.

[0297] In some instances, the methods of the present disclosure may be used to treat minimal residual disease (MRD) remaining after a prior cancer treatment. These compounds can be used to target, treat, or eradicate a subject from MRD. Treatment and / or clearance may be delayed if MRD is refractory or refractory to prior therapy. Whether or not a diagnosis has been made, it can be pursued using the present method. The methods of the present disclosure may be used in combination with prior treatments or other treatments using the multispecific antibodies described herein. After a determination that MRD is refractory to one or more available treatment options, The present invention can be used to target, treat, and / or eliminate a subject.

[0298] In certain embodiments, the antibodies herein are directed against MDR1-expressing or MDR1-overexpressing hematological cancers and It may be used in the treatment of solid tumors, including, but not limited to, one or more chemotherapeutic agents and and / or non-response or poor response to biologic agents, or resistance or tolerance In certain embodiments, the antibodies of the present invention can be used to treat cancers that are resistant to HIV-1, including ... multidrug resistant. Cancers treated with CD4+ may be targeted by other supportive stimulators such as 4-1BB (i.e., CD137), CD27, and / or CD28. The target gene is one that further expresses or overexpresses a stimulatory molecule.

[0299] Preferably, cancers that can be treated with the anti-MDR1 antibodies of the present invention further overexpress CD47. , which has been shown to play an immunomodulatory role in human malignancies and to play a role in tumor invasion and metastasis. These cells express both MDR1 and CD47, which are cell surface molecules known for their involvement in migration. The anti-MDR1 antibody used to treat cancers preferably binds to both MDR1 and CD47. It is multispecific (e.g., bispecific) with affinity.

[0300] The antibodies disclosed herein can be used as single agents (monotherapy) or in combination with other therapeutic modalities. In combination, these may be used to treat such cancers. Other treatment modalities include, for example, , a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof The treatment may include the administration of at least one additional active agent, such as a combination of chemotherapeutic agents and and / or reduction in the effective dose of biologics, reduction or avoidance of side effects, and / or intractability and other treatments, including improving treatment response in patients with chronic or drug-resistant (including multidrug-resistant) cancers. Target tumors include, but are not limited to, acute myocardial infarction (AML) and peritoneal carcinoma (PDC). Leukemias such as acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), ovarian cancer, These include colorectal cancer, gastric cancer, breast cancer, urothelial cancer, renal cancer, and lung cancer. Therefore, the administration of anti-MDR1 antibodies herein may be administered in combination with paclitaxel and / or docetaxel. Taxanes such as doxorubicin, daunorubicin, epirubicin, or idarubicin anthracyclines, especially doxorubicin, and / or vinblastine, vin Vinca alkaloids, such as cristine, vindesine, or vinorelbine, especially vinbrain This improves the patient's response to treatment with benzodiazepine or vincristine.

[0301] In some instances, the methods of the present invention can be used prophylactically to monitor For example, a subject in need thereof may have no detectable disease but have recurrent cancer (e.g., a drug or a combination of monospecific or multispecific steroids as described herein, if the individual is at risk of developing a cancer, including drug-resistant cancer. In some instances, the subject may be administered a treatment comprising one or more of the following: Particularly high risk of developing a primary cancer that is predicted to be or become drug resistant A preventative approach may be used if the subject has previously had cancer. A preventative approach if you are currently receiving treatment and are at risk of relapse or development of drug resistance can be adopted.

[0302] In some examples, the methods of the disclosure include analyzing the expression of one or more markers or therapeutic targets. For example, in some instances, the method may include analyzing the cancer from the subject. The sample is analyzed to determine whether the cancer has MDR1 above a predetermined threshold, a cancer-associated antigen above a predetermined threshold, or This may involve determining whether both are expressed.

[0303] In some instances, whether a subject is treated with an antibody of the disclosure is determined by an assessment of MDR1 expression. The results of the evaluation of cancer-associated antigen expression, or both, may depend on the results of the evaluation of cancer-associated antigen expression. In this example, if the cancer expresses MDR1 at or above a predetermined threshold, the subject is administered an anti-MDR1 antibody of the disclosure. or treated with a multispecific antibody of the present disclosure, if the cancer expresses MDR1 below a predetermined threshold. Alternatively, the subject may not be treated with an anti-MDR1 antibody but may be treated with a multispecific antibody of the present disclosure. stomach.

[0304] Use any convenient assay to analyze levels of MDR1 and / or cancer-associated antigens. These can be used, for example, in flow cytometry, nucleic acid-based assays ( These include, but are not limited to, techniques such as amplification, sequencing, cell cytometry, and immunohistochemistry. Any convenient biological sample can be used, including, for example, , cancer biopsy samples, including, but not limited to, one or more markers and / or targets A useful predetermined threshold for assessing expression is the correlation between the measured expression level and the corresponding control. The determination can be made by any convenient and appropriate method, including comparison of several In some instances, the level of MDR1 and / or cancer-associated antigens assayed in the sample A useful predetermined threshold for is the MDR1 and MDR2 activity measured in reference cells, such as healthy / normal cells. and / or the level of cancer-associated antigens.

[0305] [Manufacturing method] As summarized above, the methods of the present disclosure involve producing and / or administering the antibodies described herein. The subject antibody can be isolated by known methods, for example, by protein synthesis. They can be produced by conventional synthetic methods for the preparation of nucleotides; recombinant DNA methods; and the like.

[0306] 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 or Solid-phase polypeptide synthesis (SPPS) is a method for the chemical synthesis of antibodies of interest. is an example of a suitable method for this purpose, in which the C-terminal amino acid of the sequence is attached to an insoluble support. followed by the sequential addition of the remaining amino acids in the sequence. Various forms of SPPS can be used to synthesize antibodies of interest.

[0307] Standard recombinant methods can be used to produce the subject antibodies. For example, Nucleic acids encoding the light and heavy chain variable regions (linked to the constant region) are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains direct the expression of immunoglobulin polypeptides. The expression control sequences are operably linked to control sequences in the expression vector(s) that ensure may include, but are not limited to, a promoter (e.g., a naturally associated promoter) or heterologous promoter), signal sequence, enhancer element, and transcription termination sequence The expression control sequences are used to transform eukaryotic host cells (e.g., COS or CHO cells). or a eukaryotic promoter system in a vector that can be transfected. Once the vector is incorporated into a suitable host, the host will then be able to express the nucleotide sequence. It is maintained under conditions suitable for high level expression and antibody collection and purification.

[0308] Due to the degeneracy of the genetic code, a variety of nucleic acid sequences 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 polynucleotide. Polymerase chain reaction (PCR) mutagenesis of previously prepared variants of oligonucleotides Oligonucleotide-mediated mutagenesis can be used to generate target polypeptides. This is an example of a suitable method for preparing substitution, deletion, and insertion variants of DNA. See, e.g., Matthews et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA A. Hybridize oligonucleotides encoding the desired mutations to a single-stranded DNA template After hybridization, DNA polymerase is used to modify the Incorporating oligonucleotide primers into the target polypeptide DNA, selected modifications are An entire second complementary strand of the template is synthesized, encoding the mutation.

[0309] Suitable expression vectors are typically expressed as episomes in the host organism or as host vectors. Replicable as an integral part of chromosomal DNA. Generally, expression vectors are used to encode a desired DNA sequence. A selectable marker (e.g., ampicillin resistance, human gromycin-resistant, tetracycline-resistant, kanamycin-resistant, or neomycin-resistant) Includes:

[0310] Escherichia coli is used to clone polynucleotides encoding antibodies of interest. are examples of prokaryotic host cells that can be used for the preparation of microbial strains of Escherichia coli. Other suitable microbial hosts include: , bacilli such as Bacillus subtilis, and Salmonella, Serratia, and various Pseudom Other Enterobacteriaceae such as onas species are included.

[0311] Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, and suitable vectors include expression control sequences (e.g., It may optionally contain a sequence such as a promoter, an origin of replication, or a termination sequence. Induction of phosphoglycerate kinase and other glycolytic enzymes. Possible yeast promoters include, among others, alcohol dehydrogenase, isocytochrome C, as well as promoters from enzymes involved in maltose and galactose utilization. It can be enjoyed.

[0312] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) ) to encode a polypeptide of the invention (e.g., an immunoglobulin or fragment thereof). Polynucleotides) can also be expressed and produced. nes, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO Cell lines, various Cos cell lines, HeLa cells, HEK cells, myeloma cell lines, and transformed B cells or Hybridomas are examples of such cells. The expression vectors for these cells contain an origin of replication, a promoter, and a , and expression control sequences such as enhancers (Queen et al., Immunol. Rev. 89:49 (1986) ), as well as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription factors. It may contain necessary processing information sites such as transcription terminator sequences. Examples of current regulatory sequences are immunoglobulin genes, SV40, adenovirus, and bovine papillomavirus. Promoters derived from viruses, cytomegaloviruses, etc. Co et al., J. Immunol. 148:1149 (1992).

[0313] Once synthesized (chemically or recombinantly), whole antibodies, their dimers, The individual light and heavy chains, or other forms of the subject antibodies (e.g., scFv, etc.), may be prepared by ammonium sulfate. precipitation, affinity column, column chromatography, high performance liquid chromatography Purify according to standard procedures in the art, including high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (Generally, see Scopes, Protein Purification (Springer-Verlag, NY, (19 82). The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, At least about 85%-90% pure, at least about 90%-95% pure, or 98%-99%, or or more pure, e.g., free from contaminants such as cellular debris and macromolecules other than the antibody of interest. .

[0314] In some embodiments, the method of generating a multispecific antibody of the present disclosure includes generating candidate antibodies and Such methods may involve the use of a series of steps to screen for the presence or absence of a target protein and for its activity. Through this, we have developed a multispecific antibody that specifically binds to cells expressing both MDR1 and cancer-associated antigens. The steps of such a method may produce multispecific antibodies, or antibodies each having an MDR1-binding domain. and producing a plurality of antibodies that contain or are predicted to contain a cancer-associated antigen-binding domain. a first test cell expressing MDR1 and a cancer-associated antigen, and contacting a second cell expressing either MDR1 or a cancer-associated antigen with the antibody of contacting a first cell with a multispecific antibody or antibodies; comparing the binding to the binding of the multispecific antibody to a second cell to determine a binding specificity ratio; If the ratio exceeds a predetermined threshold, the multispecific antibody or the plurality of antibodies and one or more of the following are specific for cells that express both MDR1 and a cancer-associated antigen. When such a threshold for comparative binding is used, , the threshold can vary and can range from 1.5:1 or more, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 , 8:1, 9:1, 10:1, 20:1, 50:1, 100:1, etc.

[0315] Such methods include, for example, culturing a species, including but not limited to the cells described herein. In some instances, cells and cancers that express only MDR1 can be used. Binding of the antibody to both cells expressing the relevant antigen alone can be performed. In some instances, the method may further comprise the step of: determining whether the second cell expresses MDR1 but is cancer-associated. In the case of a tumor that does not express a cancer-associated antigen, a tumor that expresses a cancer-associated antigen but does not express MDR1 is also included. The method includes contacting the cells of the method 3 with a multispecific antibody.

[0316] In some instances, such methods may include, for example, control cells, control reagents, etc. One or more controls may be used, including but not limited to: Genes or proteins whose expression, or lack of expression, is known Useful control reagents include, but are not limited to, antibodies against known targets. For example, in some instances, a control antibody, such as a monospecific antibody, may be included. Such methods include treating the first cell, the second cell, and / or the third cell with a monospecific anti-MDR1 antibody. and a control antibody selected from a monospecific anti-cancer-associated antigen antibody. Depending on the particular method used, various other or additional controls may be included as needed. It can be used.

[0317] [kit] Aspects of the present disclosure also include kits. Kits can include, for example, antibodies, multispecific antibodies, or antibodies described herein. It may include any combination of antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. The subject kit includes the antibody, a nucleic acid encoding the antibody, or a cell containing the nucleic acid. The kit can include, for example, a therapeutic kit (e.g., a kit containing an anti-MDR1 It may comprise an antibody or a multispecific antibody and one or more additional active agents, such as, for example, a chemotherapeutic agent. (if applicable), kits for producing antibodies, kits for screening antibodies, etc. The present invention can be configured for a variety of purposes, including:

[0318] Optional components of the kit vary and can include, for example: buffers; protease inhibitors; etc. For example, if the target kit contains a target nucleic acid, the nucleic acid may contain restriction sites, multiple cloning sites, primer sites, etc. The various components may be in separate containers or may be packaged together in a container with specific compatible components. The ingredients may be pre-combined in a single container if desired.

[0319] In addition to the above components, the kit may also include any other components that can be used to practice the method. The instructions for practicing the subject method may include instructions for using the subject method. Generally, the instructions are recorded on a suitable recording medium. For example, the instructions may be recorded on a suitable recording medium such as paper or plastic. The instructions may be printed on the substrate. As such, the instructions may be included in the package insert. and in the labeling of the container of the kit or its components (i.e., the package or subpackage). In other embodiments, the instructions may be present in a suitable computer-readable medium. Removable storage media, such as compact disc read-only memory (CD-ROM), digital It exists as an electronic storage data file that resides on a versatile disc (DVD), diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but are provided in a separate package, e.g. Means are provided for obtaining instructions from a remote source via the Internet. An example of such a scenario is a web ad where instructions can be viewed and / or downloaded. As with the instructions, this means for obtaining the instructions is provided by the appropriate It is recorded on a substrate.

[0320] Non-limiting examples of embodiments of the present disclosure The above-described aspects (including embodiments) of the present subject matter may be used alone or in conjunction with one or more other aspects or Without limiting the foregoing, the present disclosure may be Certain non-limiting embodiments are provided below. As will be apparent to those of skill in the art upon reading this disclosure, Each individually numbered aspect may be used interchangeably with any preceding or succeeding individually numbered aspect. This applies to all such combinations of multiple aspects. The intent is to provide support for the integration of Various modifications and variations are possible without departing from the spirit or scope of the present invention. It will be apparent to one skilled in the art that variations and modifications can be made.

[0321] 1. An antibody that specifically binds to multidrug resistance protein 1 (MDR1), wherein the antibody is listed in Table 2. The heavy chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) of the pair of variable heavy chain (VH) and variable light chain (VL) regions of competes for binding to MDR1 with an antibody comprising light chain CDRs 1-3 (LCDRs 1-3); antibody. 2. An antibody comprising a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2, and M 2. The antibody of embodiment 1, which competes for binding to DR1. 3. Selected from the group consisting of antibodies B1.28, B1.261, B1.129, B1.225, and B1.223 listed in Table 2 2. The antibody of embodiment 1, which competes for binding to MDR1 with the selected antibody. 4. The antibody of embodiment 1, comprising the HCDRs of the VH region of an antibody described in Table 2. 5. The antibody of embodiment 1 or 2, comprising an LCDR of the VL region of an antibody described in Table 2. 6. Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 2. The antibody of embodiment 1, comprising a sex-determining region (HCDR) and a light chain CDR (LCDR). 7. An antibody molecule that specifically binds to multidrug resistance protein 1 (MDR1), The antigen-binding site is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 sex-determining regions 1–3 (HCDRs 1–3) and light chain CDRs 1–3 (LCDRs 1–3); HCDRs 1-3 of the VH region of an antibody listed in Table 2; or LCDRs 1 to 3 of the VL regions of the antibodies listed in Table 2 An antibody molecule comprising: 8. HCDRs 1-3 and LCDRs 1-3 of a pair of VH and VL regions of an antibody listed in Table 2; 8. The antibody molecule of embodiment 7. 9. The antibody of embodiment 7, comprising HCDRs 1 to 3 of the VH region of a first antibody listed in Table 2. 10. The antibody molecule of embodiment 7, comprising LCDRs 1 to 3 of the VL region of a second antibody in Table 2. 11. Embodiment 7, comprising the variable light (VL) chain and / or the variable heavy (VH) chain of an antibody listed in Table 2. The antibody molecule described in 12. (i) HCDRs 1 to 3 of the VH region of the B1.28 antibody and LCDRs 1 to 3 of the VL region of the B1.28 antibody; (ii) HCDRs 1 to 3 of the VH region of the B1.261 antibody and LCDRs 1 to 3 of the VL region of the B1.261 antibody; (iii) HCDRs 1 to 3 of the VH region of the B1.129 antibody and LCDRs 1 to 3 of the VL region of the B1.129 antibody; (iv) HCDRs 1 to 3 of the VH region of the B1.225 antibody and LCDRs 1 to 3 of the VL region of the B1.225 antibody; (v) HCDRs 1 to 3 of the VH region of the B1.188 antibody and LCDRs 1 to 3 of the VL region of the B1.188 antibody; (vi) HCDRs 1 to 3 of the VH region of the B1.27 antibody and LCDRs 1 to 3 of the VL region of the B1.27 antibody; or (vii) HCDRs 1 to 3 of the VH region of the B1.223 antibody and LCDRs 1 to 3 of the VL region of the B1.223 antibody wherein the VH and VL regions are as set forth in Table 2. The antibody molecule according to embodiment 8. 13. The antibody according to any one of aspects 7 to 12, which binds to human MDR1 and cynomolgus monkey MDR1. body molecules. 14. HCDRs 1 to 3 of the VH region of the B1.129 antibody and LCDRs 1 to 3 of the VL region of the B1.129 antibody, and V comprising HCDRs 1 to 3 of the H region and LCDRs 1 to 3 of the VL region of the B1.28 antibody, or comprising HCDRs 1 to 3 of the VL region of the B1.261 antibody 14. The antibody of embodiment 13, comprising HCDRs 1 to 3 of the VH region and LCDRs 1 to 3 of the VL region of the B1.261 antibody. molecule. 15. 15. The method according to any one of embodiments 7 to 14, wherein the method binds to loop 1 in the extracellular domain of MDR1. The antibody molecule shown. 16. HCDRs 1 to 3 of the VH region and LCDRs 1 to 3 of the VL region of the B1.129 antibody, or the VH region of the B1.223 antibody and LCDRs 1 to 3 of the VL region. 17. Calcein, daunorubicin (Dioc2), and rhodamine by cancer cells The antibody molecule according to any one of aspects 7 to 16, which inhibits the above-mentioned excretion. 18. 18. The antibody molecule of aspect 17, wherein the cancer cells are 293T cells that overexpress MDR1. 19. 18. The antibody molecule of aspect 17, wherein the cancer cell is a MES-SA-DX5 cancer cell. 20. 21. Any of embodiments 17 to 20, comprising HCDRs 1 to 3 of the VH region and LCDRs 1 to 3 of the VL region of the B1.129 antibody. An antibody molecule described in any one of claims 1 to 4. twenty one. An antibody molecule that specifically binds to MDR1 expressed on a cell surface, comprising any of the following: antibody molecules, including: (i) a heavy chain complementarity-determining region (HCDR) 1 comprising the amino acid sequence GFTFSRY (SEQ ID NO: 343); HCDR2 containing the amino acid sequence SSGGGN (SEQ ID NO: 344); and a variable heavy (VH) chain comprising an HCDR3 comprising the amino acid sequence RSSQNIVHSTGNTYLD (sequence light chain complementarity determining region (LCDR) 1 containing the amino acid sequence KVSNRFS (SEQ ID NO: 12); and LCDR3 comprising the amino acid sequence FQGSHIPRT (SEQ ID NO: 13). L) chain; (ii) Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 287) a VH chain comprising the amino acid sequence DVVLTQSPLSLPVTLGQPASISCRSSQNIVHSTGNTYLDWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYFCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 20) a VL chain comprising: (iii) Amino acid sequence EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVL DSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 309) a heavy chain comprising the amino acid sequence DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 21) light chains containing; (iv) HCDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO: 346); and HCDR2 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308); and HCDR3 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308), a VH chain; and an LCDR1 comprising the amino acid sequence RSSQNIVHSTGNTYLD (SEQ ID NO: 11); LCDR2 containing the sequence KVSNRFS (SEQ ID NO: 12); and LCDR3 containing the amino acid sequence FQGSHIPRT (SEQ ID NO: 13). a VL chain comprising an LCDR3; (v) Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) VH chain and amino acid sequence comprising DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 21) light chains containing; (vi) Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 310) heavy chain and amino acid sequence comprising DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 21) light chains containing; (vii) HCDR1 comprising the amino acid sequence GFTFSRY (SEQ ID NO: 343); and and HCDR2 comprising the amino acid sequence GAGDAWFAY (SEQ ID NO: 345). a VH chain; and an LCDR1 comprising the amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 290); LCDR2 containing the amino acid sequence KVSNRFS (SEQ ID NO: 12); and the amino acid sequence QGSHFPRT (SEQ ID NO: 348). a VL chain comprising an LCDR3 comprising: (viii) Amino acid sequence evkvvesggvlvrpggslklscaasgftfsrytmswvrqtpekrlewvatissgggntyypdsvkgrftvsrdnamssly lqmsslrsedtalyycarygagdawfaywgqgtlvtvss (SEQ ID NO: 287) and a VH chain comprising the amino acid sequence DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) a VL chain comprising: (ix) Amino acid sequence EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVL DSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 309) heavy chain and amino acid sequence comprising DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) light chains containing; (x) HCDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO: 346); and HCDR2 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308); and HCDR3 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308), a VH chain; and an LCDR1 comprising the amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 290); LCDR2 containing the sequence KVSNRFS (SEQ ID NO: 12); and LCDR3 containing the amino acid sequence QGSHFPRT (SEQ ID NO: 348). a VL chain comprising an LCDR3; (xi) Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY VH chain and amino acid sequence comprising MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) light chains containing; (xii) amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 310) heavy chain and amino acid sequence comprising DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) light chains containing; (xiii) HCDR1 containing the amino acid sequence GFTFSRY (SEQ ID NO: 343); and and HCDR2 comprising the amino acid sequence GAGDAWFAY (SEQ ID NO: 345). a VH chain; and an LCDR1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 39); LCDR2 containing the amino acid sequence KVSKRFS (SEQ ID NO: 40); and the amino acid sequence FQASHFPRT (SEQ ID NO: 329). a VL chain comprising an LCDR3 comprising: (xiv) Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 287) and a VH chain comprising the amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) a VL chain comprising: (xv) amino acid sequence EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVL DSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 309) heavy chain and amino acid sequence comprising DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) a VL chain comprising: (xvi) HCDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO: 346); and HCDR2 containing the amino acid sequence GGYRAMDY (SEQ ID NO: 308); and HCDR3 containing the amino acid sequence GGYRAMDY (SEQ ID NO: 308). , a VH chain; and an LCDR1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 39); LCDR2 containing the amino acid sequence KVSKRFS (SEQ ID NO: 40); and LCDR3 containing the amino acid sequence FQASHFPRT (SEQ ID NO: 329). a VL chain comprising LCDR3 and a VL chain comprising CDR 5F9 / VL B1-89v1 (xvii) amino acid sequence qvqlvqsgaevkkpgasvkvsckasgytftnynmhwvrqapgqrlewmgtiypgnddtsynqkfkdrvtitadtsastay melsslrsedtavyycarggyramdywgqgtlvtvss (SEQ ID NO: 294) VH chain and amino acid sequence comprising DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) a light chain comprising: (xviii) amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 310) heavy chain and amino acid sequence comprising DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) A light chain comprising: twenty two. A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and tumor-associated antigens (TAA) and a second VH chain, each of which is a nucleotide sequence of a first VH chain and a second VL chain. wherein each of the VL chains comprises an antigen-binding site for MDR1, and the first VH chain comprises an antigen-binding site for MDR 1, and the second VH chain comprises an antigen-binding site for TAA. wherein said second VH chain binds to a TAA when paired with one of said light chains. . twenty three. The antigen-binding sites of the two VL chains correspond to light chain CDRs 1 to 3 (LCDRs 1 to 3) of the antibodies listed in Table 2. 23. The bispecific antibody molecule of embodiment 22, comprising: twenty four. the antigen-binding site of the first VH chain is selected from the heavy chain CDRs 1 to 3 (HCDRs 1 to 3) of an antibody listed in Table 2 24. The bispecific antibody molecule of embodiment 22 or 23, comprising:

[0322] twenty five. the first VH chain comprises HCDRs 1 to 3 of an anti-MDR1 antibody other than an antibody listed in Table 2, and optionally wherein the first VH chain comprises the amino acid sequence of the VH chain of an anti-MDR1 antibody other than the antibodies listed in Table 2. 24. A bispecific antibody molecule according to embodiment 22 or 23. 26. The bispecific antibody molecule of embodiment 25, wherein the anti-MDR1 antibody comprises: Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO: 293) the VH chain of the 15D3 antibody having the formula: Amino acid sequence AVQLQQSGPELVKTGASVKISCKASGYSFSNYYIHWVKQSHGKSLEWIGFISCYNGATFYNQKFKGKATFTVDTSSSTAY MKFNSLTFEDSAVYYCARLPIQFGNFYPMDYWGQGTSVTVSS (SEQ ID NO: 288) or a VH chain of a UIC2 antibody having Amino acid sequence EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLY LQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA (SEQ ID NO: 286) The VH chain of the MRK16 antibody having the following structure: 27. the anti-MDR1 antibody has the amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 292) wherein X comprises a VH chain having 2 is N, Q or S, and optionally X 2 is Q or S, 27. The bispecific antibody molecule according to 26. 28. the VL chain comprises LCDRs 1 to 3 of the B1-28 antibody shown in Table 2; Optionally, the VL chain comprises the amino acid sequence of the VL chain of the B1-28 antibody listed in Table 2; or , The VL chain has the amino acid sequence LWVPGSTGDVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS Anti-MDR1 with a VL chain comprising GTDFTLKISRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 289) comprising LCDRs 1 to 3 of the V6 antibody; Optionally, the VL chain comprises the amino acid sequence of the VL chain of the V6 antibody. A bispecific antibody molecule according to any one of aspects 22 to 27. 29. the VL chain comprises LCDRs 1-3 of an anti-MDR1 antibody other than an antibody listed in Table 2, and optionally the light chain comprises: 25. The antibody of claim 22 or 24, comprising the amino acid sequence of the VL chain of an antibody other than an antibody listed in Table 2. The bispecific antibody molecule described above. 30. the anti-MDR1 antibody Amino acid sequence DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 283) the variable light chain of the MRK16 antibody comprising: Amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 284) the variable light chain of the 15D3 antibody comprising: Amino acid sequence DVVMTQTPRSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHIPPWTFGGGTKLDIK (SEQ ID NO: 285) The variable light chain of a UIC2 antibody comprising 30. The bispecific antibody molecule of embodiment 29, comprising LCDRs 1 to 3 of: 31. the antibody inhibits MDR1-mediated efflux when bound to cells expressing MDR1; 1. The antibody molecule or bispecific antibody molecule of any one of embodiments. 32. the antibody is capable of increasing the sensitivity of cancer cells to treatment with a chemotherapeutic agent; wherein the half maximal inhibitory concentration (IC50) of the chemotherapeutic agent when co-administered with the antibody is: The following array EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO: 293) and a VH chain having The following array DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 284) VL chain having at least 5-fold lower than the IC50 of the chemotherapeutic agent when co-administered with an anti-MDR1 antibody comprising A bispecific antibody molecule according to any one of aspects 22 to 31. 33. 33. The bispecific antibody molecule of any one of aspects 22 to 32, wherein the TAA is CD47. 34. The bispecific antibody comprises HCDRs 1 to 3 of the VH region of the anti-CD47 antibody 5F9, and the VH region comprises the following: The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) or The bispecific antibody comprises HCDRs 1 to 3 of the VH region of the anti-CD47 antibody B6H12, and the VH region is The amino acid sequence below EVQLVESGGDLVKPGGSLKLSCAASGTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNTLY LQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSS (SEQ ID NO: 295) 34. The bispecific antibody molecule of embodiment 33, comprising: 35. the first VH chain comprises HCDRs 1 to 3 of a VH chain derived from an anti-MDR1 antibody having the following HCDR sequences: HCDR1: RYTMS (SEQ ID NO: 301), HCDR2: TISSGGGNTYYPDSVKG (SEQ ID NO: 302), TISSGGGQTY YPDSVKG (SEQ ID NO: 303), or TISSGGGSTYYPDSVKG (SEQ ID NO: 304), and HCDR3: ARY GAGDAWFAY (SEQ ID NO: 349); The VL chain comprises LCDRs 1 to 3 of the VL region of the B1-28 antibody, respectively, as set forth in Table 2, wherein LCDR The R sequences are LCDR1: RSSQNIVHSTGNTYLD (SEQ ID NO: 11), LCDR2: KVSNRFS (SEQ ID NO: 12), and and LCDR3: FQGSHIPRT (SEQ ID NO: 13), 35. A bispecific antibody molecule according to embodiment 34. 36. the first VH chain EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX2TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 291) wherein X2 is N, Q, or S; the second VH chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) and an amino acid sequence comprising: The two VL chains comprise amino acid sequences comprising the VL region chains of the B1-28 antibody set forth in Table 2. 36. A bispecific antibody molecule according to embodiment 35. 37. The first VH chain is selected from the group consisting of (i) B1.28 antibody; (ii) B1.30 antibody; (iii) B1.89 antibody; and (v) HCDRs 1 to 3 of the VH chain of the B1.129 antibody; (v) B1.225; (vi) B1.261 antibody; or 15D3 or comprises HCDRs 1 to 3 of the VH chain of the MRK16 antibody; The identical VL chains each comprise (i) LCDRs 1 to 3 of the VL chain of the MRK16 antibody, or (ii) LCDRs 1 to 3 of the VL chain of the V6 antibody. comprising LCDRs 1 to 3 of the VL chain; and the second VH chain comprises HCDRs 1 to 3 of the VH chain of the 5F9 antibody or the B6H12 antibody; 23. A bispecific antibody molecule according to embodiment 22. 38. The first VH chain is selected from the group consisting of (i) B1.188 antibody; (ii) B1.28 antibody; and (iii) B1.261 antibody, as listed in Table 2. (iv) B1.129 antibody; or (v) B1.223 antibody VH chain HCDRs 1 to 3, and the same VL chain each comprising LCDRs 1 to 3 of the VL chain of the MRK16 antibody; and the second VH chain comprises LCDRs 1 to 3 of the 5F9 antibody or B6H12 antibody. 23. A bispecific antibody molecule according to embodiment 22, comprising HCDRs 1 to 3 of the VH chain of said antibody. 39. The first VH chain is selected from the group consisting of (i) B1.188 antibody; (ii) B1.28 antibody; and (iii) B1.261 antibody, as listed in Table 2. (iv) the VH chain of the B1.129 antibody; or (v) the VL chain of the B1.223 antibody, each of which is an MRK1 38. The method of claim 37, wherein the first VH chain comprises the VL chain of the 5F9 antibody or the B6H12 antibody. A bispecific antibody molecule according to claim 1. 40. the first VH chain comprises HCDRs 1 to 3 of the VH chain of the B1.225 antibody listed in Table 2, and the same VL chain each comprising LCDRs 1 to 3 of the VL chain of the MRK16 antibody, and the second VH chain comprises LCDRs 1 to 3 of the 5F9 antibody or B6H12 28. A bispecific antibody molecule according to embodiment 27, comprising HCDRs 1 to 3 of the VH chain of said antibody. 41. the first VH chain comprises the VH chain of the B1.225 antibody listed in Table 2, and the identical VL chain comprises the VL chain of the B1.225 antibody listed in Table 2, 41. The dual antibody of claim 40, wherein said first VH chain comprises the VL chain of a K16 antibody, and said second VH chain comprises the VH chain of a 5F9 antibody. Isomeric antibody molecule. 42. 10. The antibody molecule or bispecific antibody of any preceding aspect, wherein the antibody comprises a humanized light chain. antibody molecule. 43. 10. The antibody molecule or bispecific antibody of any preceding aspect, wherein the antibody comprises a humanized heavy chain. antibody molecule. 44. 2. The method of claim 1, wherein the compound of claim 1 is a compound selected from the group consisting of benzodiazepines, ... wherein the method comprises administering the antibody to the subject. An antibody molecule or a bispecific antibody molecule, comprising: 45. The method 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 45. The antibody molecule or bilayer according to embodiment 44, comprising an antibody molecule or bilayer comprising an antibody inhibitor, an immunotherapeutic agent, or a combination thereof. Heavy specific antibody molecule. 46. The at least one additional active agent is a chemotherapeutic agent, and optionally, the chemotherapeutic agent is a 46. ​​The anti-cancer agent of embodiment 45, which is a quinol, a vinca alkaloid, or an anthracycline. antibody molecule or bispecific antibody molecule. 47. the subject to be treated has a cancer that has been determined to be resistant to treatment with said chemotherapeutic agent; 47. An antibody molecule or bispecific antibody molecule according to embodiment 46. 48. The antibody molecule according to any one of aspects 1 to 21 or any one of aspects 22 to 43. and administering to the subject a therapeutically effective amount of the bispecific antibody molecule described herein. How to treat. 49. The method comprises administering to said patient an antibody molecule or bispecific antibody molecule in combination with at least one additional active agent. and administering an isomeric antibody molecule to the subject, wherein the at least one additional active agent is a chemotherapeutic agent. , an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. 48. The method according to claim 48.

[0323] 50. The at least one additional active agent is a chemotherapeutic agent, and optionally, the chemotherapeutic agent is a 50. The method of claim 49, wherein the compound is a benzodiazepine, a vinca alkaloid, or an anthracycline. Law. 51. an embodiment in which the subject being treated has a cancer that is classified as resistant to said chemotherapeutic agent; 50. The method according to claim 50. 52. The antibody molecule according to any one of aspects 1 to 21 or any one of aspects 22 to 43. One or more nucleic acids comprising one or more sequences encoding the bispecific antibody molecule described. 53. one or more recombinant expression vectors comprising one or more nucleic acids according to embodiment 52; . 54. A host cell genetically modified with one or more recombinant expression vectors according to embodiment 52. . 55. Chimeras containing MDR1-binding domains, transmembrane domains, and intracellular signaling domains an immune effector cell comprising an antigen receptor (CAR), wherein the MDR1-binding domain is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 and an immune effector cell, comprising a light chain CDR (LCDR) and / or a light chain CDR (HCDR). 56. A method for inhibiting the efflux activity of MDR1 expressed by a living cell, comprising: The antibody molecule according to any one of aspects 1 to 21 or any one of aspects 22 to 43 a bispecific antibody molecule. 57. 57. The method of embodiment 56, further comprising contacting the cell with an inhibitor of MDR1-mediated efflux. . 58. 58. The method of embodiment 56 or 57, further comprising contacting the cells with a chemotherapeutic agent. Law. 59. 59. The method of any one of aspects 56 to 58, wherein the cells are cancer cells. 60. The method of embodiment 59, wherein the cancer cells are multidrug resistant cancer cells. 61. A method for assaying expression of MDR1 on the cell surface of a cell, comprising: 8. A method comprising contacting a subject with an antibody molecule described in any one of claims 7. 62. 62. The method of embodiment 61, wherein the antibody is detectably labeled. 63. 1. A method for diagnosing a disease or condition associated with MDR1 expression in a subject, comprising: A tissue or cell sample from a subject is subjected to an immunoprecipitation of the antibody molecule according to any one of aspects 1 to 47. contacting said mixture. 64. The antibody molecule has the VH and VL chain configuration shown for the antibody molecule in FIG. 64. The method of embodiment 63, comprising: 65. An antibody that specifically binds to multidrug resistance protein 1 (MDR1) for use as a medicine. The antibody variable heavy chain (VH) and variable light chain (VL) regions of the antibody shown in Table 2 are overlapping. an antibody comprising light chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) and light chain CDRs 1 to 3 (LCDRs 1 to 3); An antibody that competes for binding to R1. 66. Specific binding to multidrug resistance protein 1 (MDR1) for the manufacture of a medicament for the treatment of cancer Use of an antibody, said antibody comprising a variable heavy chain (VH) region and a variable heavy chain (VH) region of an antibody listed in Table 2. The variable light chain (VL) region pair comprises heavy chain complementarity determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3). 1 to 3), which competes for binding to MDR1 with an antibody comprising the compound. 67. Multidrug resistance protein 1 (MDR1) for use in a method of treating cancer in a subject 1. A pharmaceutical composition comprising an antibody that specifically binds to a nucleotide sequence of a nucleotide sequence selected from the group consisting of nucleotides 1 and 2, wherein the antibody is an antibody listed in Table 2. The heavy chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) of the pair of variable heavy chain (VH) and variable light chain (VL) regions of It competes for binding to MDR1 with an antibody comprising light chain CDRs 1-3 (LCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3), The method comprises administering the antibody to the subject. 68. Antibody molecules that specifically bind to multidrug resistance protein 1 (MDR1) for pharmaceutical use The antigen-binding site of the antibody molecule is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 sex-determining regions 1–3 (HCDRs 1–3) and light chain CDRs 1–3 (LCDRs 1–3); HCDRs 1-3 of the VH region of an antibody listed in Table 2; or LCDRs 1 to 3 of the VL regions of the antibodies listed in Table 2 Including, antibody molecule. 69. Specific binding to multidrug resistance protein 1 (MDR1) for the manufacture of a medicament for the treatment of cancer Use of an antibody molecule, wherein the antigen-binding site of said antibody molecule comprises: Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 Sex-determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3); antibodies listed in Table 2 or LCDRs 1 to 3 of the VL region of an antibody listed in Table 2, use. 70. Multidrug resistance protein 1 (MDR1) for use in a method of treating cancer in a subject a pharmaceutical composition comprising an antibody that specifically binds to a target polypeptide, wherein the antigen-binding site of the antibody molecule is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 Sex-determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3); antibodies listed in Table 2 or LCDRs 1 to 3 of the VL region of an antibody listed in Table 2, The method includes administering the antibody to the subject. Pharmaceutical compositions. 71. 22. The antibody molecule of embodiment 21 for use as a medicament. 72. 22. Use of an antibody molecule according to embodiment 21 for the manufacture of a medicament for the treatment of cancer. 73. 22. A method for treating cancer in a subject, comprising administering to a subject a therapeutically effective amount of an antibody molecule according to embodiment 21. a pharmaceutical composition comprising the antibody, the method comprising administering the antibody to the subject. Finished product. 74. Multidrug resistance protein 1 (MDR1) and tumor-associated antigens (TAA) for pharmaceutical use a bispecific antibody molecule that binds to a first variable heavy (VH) chain and a second variable light (VL) chain; ) chain, and a second VH chain, each of the VL chains comprising an antigen-binding site for MDR1; The first VH chain comprises an antigen-binding site for MDR1, and the second VH chain comprises an antigen-binding site for TAA. and wherein the second VH chain binds to the TAA when paired with one of the light chains. A bispecific antibody molecule. 75. Multidrug resistance protein 1 (MDR1) and tumor-associated antigens for the manufacture of medicaments for the treatment of cancer The use of a bispecific antibody molecule that binds to a TAA, said antibody molecule binding to two identical potential TAAs. a variable light (VL) chain, a first variable heavy (VH) chain, and a second VH chain, each of the VL chains being an MDR1 the first VH chain comprises an antigen-binding site for MDR1, The second VH chain comprises an antigen-binding site for the TAA, and the second VH chain binds to one of the light chains. The use of a nucleotide sequence that binds to the TAA when paired with one of the nucleotide sequences. 76. Multidrug resistance protein 1 (MDR1) for use in a method of treating cancer in a subject and a pharmaceutical composition comprising a bispecific antibody molecule that binds to a tumor-associated antigen (TAA), The antibody molecule comprises two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain. wherein each of the VL chains comprises an antigen-binding site for MDR1, and the first VH chain comprises an antigen-binding site for MDR1. the second VH chain comprises an antigen-binding site for said TAA, a second VH chain that binds to the TAA when paired with one of the light chains, and the method further comprises: A pharmaceutical composition comprising administering the antibody to the subject. 77. 75. The bispecific molecule according to embodiment 74, wherein the bispecific molecule comprises LCDRs 1 to 3 of the VL region of an antibody listed in Table 2. 77. A bispecific molecule according to embodiment 75, a use according to embodiment 75, or a pharmaceutical composition according to embodiment 76.

[0324] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0325] The following examples will provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. are described to provide and limit the scope of what the inventors regard as their invention It is not intended to be an exhaustive list of experiments, nor is it intended that the experiments below represent all or the only experiments performed. The accuracy of any values ​​used (e.g., amounts, temperatures, etc.) is not intended to be accurate. Efforts have been made to ensure, but some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, and temperatures are in °C. and the pressure is at or near atmospheric pressure.

[0326] General methods for molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Mole cular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Me thods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Pre ss 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998) The disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for the method were purchased from BioRad, Agilent Technologies, and chnologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), and from distributors such as Addgene, Inc., American Tycoon, Inc. These are available from repositories such as the American College of Tissue Culture Collection (ATCC).

[0327] Example 1: Specific binding to cells expressing high levels of Pgp (MDR1), but not MDR1 expression production of antibodies that bind significantly less or not at all to cells with reduced or absent

[0328] Materials and Methods antibody production For immunization, wild-type (WT) and mutant MDR1 were used. a mutant MDR1 containing a natural mutation, and another MDR1 containing a mutation that locks the pump in a closed configuration Mutants were generated in human and Macaca f to generate MDR1 mutants constrained in the open conformation. The mutations in MDR1 of C. ascicularis are E556Q and E1201Q. The mutations in human MDR1 to generate the MDR1 mutants are as follows: (i) K4 33M, S434A, K1076M, S1077A; (ii) K433M, S434A, Q475A, K1076M, S1077A, Q1118A; and / or (iii) K433M, S434A, Q475A, R588E, K1076M, S1077A, Q1118A, R1233E. Additional MDR1 mutants are homologous to human MDR1, with deletions of amino acid residues 82-99 or 79-102. The amino acid residue numbering for the substituted positions is: Numbering refers to the sequence of human MDR1.

[0329] Wild-type (WT) and mutant MDR1 were expressed in 3T3 cells. MDR1 (wild-type or mutant) DNA and / or MDR1 (wild-type or mutant) DNA were administered for 8 weeks using the Female mice were immunized with cells expressing the IgG1 or IgG2 mutant. In some cases, the number of antibodies produced was To increase diversity, multiple antigens (one or more of WT or mutant MDR1) were used in the immunization. Spleen and lymph node cells were fused with SP2 / 0 myeloma cells. Cytometry was performed to detect the presence of anti-ABCB1 antibodies, followed by their function in cell killing assays. The hybridomas were screened for activity. CDRs from mouse IgG were cloned into a mammalian IgG1 backbone expression vector and expressed in HEK 293 mammals. The antibody was transfected into a human cell line to express the full-length IgG1 antibody.

[0330] DNA sequencing The DNA sequence was determined by double-strand sequencing.

[0331] DNA and protein sequence analysis and sequence data management The Vector NTI (ThermoFisher) software package was used for sequence mapping, analysis, and Used for annotation and illustration purposes.

[0332] Cell culture and antibody production Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harfor d, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, I Standard cell culture techniques are used as described in nc.

[0333] Expression vector For the generation of antibody expression vectors, the variable regions of the heavy and light chain DNA sequences are cloned into mammalian cells. The corresponding universal recipient expression vectors were previously optimized for expression in cell lines. In-frame with either the inserted human IgG1 constant heavy chain or the human IgG1 kappa constant light chain The gene to be expressed was subcloned into the full-length human HIV-1 vector for high-level gene expression. The pCI-neo mammalian expression vector uses the cytomegalovirus (CMV) immediate early promoter. The two antibody chains were cloned into two different vectors. I liked it.

[0334] The N-terminal signal sequences from mouse IgG heavy chain and κ light chain were used for secretory expression of the heavy and light chains, respectively. The signal peptide was cleaved during expression, leaving an intact N-terminus. In this product, the C-terminus of the CH1 IgG1 constant region was fused to a 6xHis tag for purification.

[0335] Monoclonal antibodies raised against Pgp were expressed in human IgG1 / κ as recombinant antibodies. It was cloned into a vector.

[0336] Variable heavy and light chain fragments were obtained from mouse hybridoma sequences and the leader sequence and constant regions were cloned into the same background.

[0337] Production of mAb, Fab'2, Fab and bispecific mAb Expi293 cells (A14527, Thermo Fisher Scientific) grown in suspension were cultured according to the manufacturer's recommendations. Using polymer-based co-transfection of mammalian expression vectors in guinea pig cells , antibody constructs were expressed.

[0338] Approximately 6 days after transfection, the cells were harvested by centrifugation. 1 μg of total coding DNA per ml of transfected culture was added to Opti-MEM® medium (Life Technologies) in the same medium. The mixture was then incubated with PBS (pH 7.5) for 20 minutes at 37°C under an 8% CO2 atmosphere in air. The cells were then dispersed in Expi293® Expression Medium (Life Technologies) at 2.5 million cells / ml in suspension. Six days later, the medium containing the antibody construct was added to Expi 293® cells growing in the presence of IgG. was collected by centrifugation.

[0339] mAb purification For purification of Fc-containing antibody formats, use 10 μl of MabSelect per ml of supernatant. TM SuRe TM (GE Healthcare) was added to the harvested medium and stirred overnight at 4°C. An empty manifold unit (Pall Lifesciences, USA) was used to measure the 24-well filter plate. Protein A resin was applied in a 500-well plate. The resin was washed with PBS and the antibody was added in 50 mM phosphate pH 3. and neutralized with 10x PBS pH 13.

[0340] Fab was purified using Ni Sepharose 6 Fast Flow Histidine-tagged Protein Purification Resin (GE Healthcare) The beads were purified according to the same procedure using 20 mM imidazole after washing with PBS. The complex was washed with 25 mM phosphate buffer pH 7.4, 150 mM NaCl supplemented with 2 volumes of HCl. Eluted with 25 mM phosphate buffer, pH 7.4, 150 mM NaCl supplemented with 500 mM imidazole. Finally, the purified Fab was buffer exchanged into PBS. The ram is shown in Figure 1.

[0341] mAb analytical testing (GXII reduced and non-reduced) The purity and monomer content of the final protein preparation are as described by the manufacturer. Caliper's LabChip GX using the Protein Express LabChip Kit (Perkin-Elmer) as shown. The results were determined by high-throughput analysis using a HPLC-based HPLC system. The chips were incubated in 0.2% SDS and 1% CO₂ on the instrument. The destaining channel was automatically primed with a polymer solution containing a fluorescent dye. The polymer solution was filled with S and dye-free polymer solution. Briefly, the polymer solution was filled with or without DDT. By mixing a small amount of sample (2-5 μL) with caliper sample buffer, the reduction and Protein samples were denatured at 75°C for 5 minutes and centrifuged at 2000 g for 3 minutes. The electrophoresis was performed using LabChip GXII Touch software (Perkin Elmer). A gas electrophoresis diagram was prepared.

[0342] Reagents used to test binding, affinity, efflux blockade, and cellular sensitization to chemotherapeutic agents cell line Human embryonic kidney (HEK) cell line HEK 293FT (Life Technologies) was cultured in 10% fetal bovine serum (Hy) medium. Clone), 2 mM GlutaMAX (Life Technologies), 100 U / mL penicillin, and 100 μg / mL The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with streptomycin at 37°C in 5% CO2. Maintained in incubation.

[0343] Optimized PEIPro TM Using the transfection protocol (Polyplus), pLen 293T cells were cultured with a human P-glycoprotein tagged ORF clone in the ti-C-Myc-DDK-P2A-Puro plasmid. DNA and JetPEI® were added to the culture medium at 100°C for 1 hour. After dilution with 10 ml of PBS, the mixture was gently mixed for approximately 10 minutes. The efflux blockade was performed according to the manufacturer's protocol. Multidrug resistance was measured using the Multidrug Resistance Direct Dye Exclusion Assay (Chemicon) according to the protocol.

[0344] Detection of ABCB1-specific binding The binding specificity of the mAb, Fab, and Fab'2 antibodies was confirmed in the 293T cell line, which naturally expresses Pgp, and human Pgp 293T naive cells overexpressing the target, Pgp knockdown 293T cells (lentiviral iRNA) After treatment, the cells were examined by FACS. Amounts of mAb or bispecific mAb, or human IgG1 isotype control antibody were used. The cells were incubated on ice for 1 hour. The cells were then washed three times with FACS buffer (PBS containing 0.5% BSA). Alexa647-labeled goat anti-human antibody was added as a secondary antibody, and the samples were incubated on ice for another 1 The samples were washed and analyzed using a BD FACSCanto (BD Biosciences). did.

[0345] Generation of MDR1-overexpressing cell lines After transfection with human MDR1 cDNA in a mammalian expression vector, 0.2 m Transfectants for resistance to hygromycin at 0.5 mg / ml or 0.5 mg / ml By multi-step selection, MDR1-overexpressing HEK 293T cells or C6 cells were isolated. After incubation with the detection antibody MRK16-PE, high MDR1 expression was detected using FACS Aria. A clone was isolated.

[0346] Emission interception experiment procedure HEK 293T cells expressing human MDR1 or MES-SA / DX5 cells expressing human MDR1 were washed several times. and plate at a cell density of 1 x 10e6 cells / ml in phenol red-free DMEM in a 96-well plate. The cells were mixed with 300 nM to 1 nM of antibody and incubated for 37 min in 50 μl aliquots per well. The cells were incubated at 4°C for 1 hour. To measure efflux blockade, cells were incubated with B1 fluorophores as follows: Incubated in the presence of photosubstrates: 0.6 μM rhodamine; 5 μM daunorubicin (DiOC2( 3)) or 0.6 μM calcein AM for 1.5 hours at 37°C. The cells were then washed twice and finally The cells were resuspended in 1 ml PBS. Fluorescence of rhodamine, DiOC2(3), or calcein AM was measured using a flow cytometer. was measured by spectrometry.

[0347] Cell binding assay Antibody binding to cells was assessed by flow cytometry. 293T cells (293T_KPB1_OX) stably transfected with 293T were analyzed by flow cytometry. Wash once in buffer (PBS + 2% FBS + 0.02% sodium azide) and analyze for flow cytometry. Resuspend cells at 2 x 10^6 cells / mL in buffer and plate at 0.1 mL / well in a 96-well microtiter plate. In the figure, "B1" represents ABCB1 (also known as MDR1 or Pgp). Antibodies were added to cells at 5 μg / mL for initial binding confirmation or analyzed by flow cytometry. Serial dilutions were made in buffer starting from 100 μg / mL. Cells were incubated on ice for 30 minutes. Afterwards, the cells were washed twice with flow cytometry buffer. The bound antibodies were purified by PE-labeled F(ab')2 fragments. Detection was performed with goat anti-human IgG (Jackson ImmunoResearch) and analyzed using an Attune NxT flow cytometer. Evaluated above.

[0348] Titration of monoclonal antibodies that bind to KPB1 The binding titration of the recombinant antibody to the ABCB1 transfectant was 666.7 nM The assay was performed by three-fold serial dilutions of antibodies from 1000-2000 diluted in flow cytometry buffer. The antibodies were incubated with the cells for 30 minutes on ice. The cells were washed twice with flow cytometry buffer. After washing, the bound antibody was purified by centrifugation with PE-labeled Fab (ab) at a 1:200 dilution in flow cytometry buffer. The antibodies were detected with 2-fragment goat anti-human IgG (Jackson ImmunoResearch) and incubated with the cells for 20 minutes on ice. After washing twice with flow cytometry buffer, the cells were incubated in the Attune NxT flow cytometer. Fluorescence was measured using a fluorimeter. Data were analyzed using GraphPad Prism 8.0 software to calculate EC50 values. It was decided that:

[0349] Cytotoxicity assay The effect of antibodies against vincristine cytotoxicity was investigated in human acute lymphoblastic leukemia (ALL). The doxorubicin-selected ABCB1-positive variant of the NALM6 cell line, N6 / ADR, was evaluated. In a white flat-bottom 96-well tissue culture plate, 5000 cells / well were cultured in 0.05 mL of uptake. Cells were plated in Seiyaku medium (RPMI-1640 + 10% FBS). Test or control antibodies at 7 μM (2× final concentration) or a small molecule KPB1 inhibitor at 7 μM (2× final concentration) Vincristine was tested by serial dilution from 200 μM in assay medium containing valspodar. Vincristine was prepared at 2x the final assay concentration. An equal volume (0.05 mL) of vincristine / antibody mixture was added. was added to N6 / ADR cells in a 96-well plate. The plate was then incubated at 37°C in 5% CO After 72 hours, the plates were equilibrated to room temperature and incubated according to the manufacturer's recommended protocol. CellTiter-Glo® Luminescent Cell Viability Assay was used according to the protocol. Luminescence was measured using a Molecular Devices® FlexStation®. The data were analyzed on a standard multimode microplate reader and analyzed using GraphPad Prism 8.0. The analysis was carried out using software.

[0350] Example 2: In vivo efficacy of anti-ABCB1 monoclonal antibodies Anti-ABCB1 antibodies B1.129, B1.261, B1.223, B1.225, B1.188, B1.28, and B1.226 were detected in immunocompetent individuals. The effect on tumor growth in all mice was tested. The following cancer cell lines were used:

[0351] MES-SA DX5: The multidrug-resistant cell line MES-SA / DX5 (Sigma. 95051031) was originally derived from a hysterectomized 56-year-old Caucasian woman. The human uterine sarcoma cell line MES-SA (Sigma catalog no. 95051030) was derived from a tumor obtained at the time of the study. This DX5 variant is 100-fold more resistant to doxorubicin, The doubling time is reported to be 30 hours. MES-SA / Dx-5 cells are resistant to many chemotherapeutic drugs, including daunol. (e.g., bicine, dactinomycin, vincristine, taxol, colchicine) It exhibits marked cross-resistance and moderate cross-resistance to mitomycin C and melphalan. Bleomycin, cisplatin, carmustine, 5-fluorouracil or methotrexate No cross-resistance to Rexate was observed.

[0352] A2780 ADR: The adriamycin-resistant cell line A2780ADR was derived from the parental A2780 cell line (Sigma catalog no. 931125 19) was developed by exposing it to adriamycin. A2780ADR was developed by exposing it to melphalan and To maintain resistance, the medium should be supplemented with Adriamycin. The cells grow in monolayer and spinner suspension cultures. It is tumorigenic in immunodeficient mice. Together, these strains differ only in exposure to a single drug, highlighting the role of pleiotropic drugs in human ovarian cancer. This will facilitate the exploration of molecular changes involved in the development of antibiotic resistance.

[0353] MES-SA DX5 and A2780 ADR cells express the efflux pump ABCB1 and the tumor target CD47. The bispecific antibody (KB1-1401) containing 15D3VH:MRK16VL::MRK16VL:5F9VH chains was synthesized using MES-SA DX5 and The KB1-1401 bispecific antibody exhibits binding to both A2780ADR and A2780ADR in in vivo studies. , MES-SA DX5 and A2780ADR tumors to paclitaxel. The antibody is also called KNJY Bis P1.1 or KBis1.1.

[0354] ovarian carcinoma Ovarian carcinoma was modeled by implanting A2780 ADR cells into nude mice. Monoclonal antibodies are efficacious in the A2780 ADR ovarian carcinoma model in nude mice Anti-ABCB1 antibodies B1.28, B1.261, B1.129, B1.226, B1.223, B1.225, and B1.188 are available as single agents. In combination with paclitaxel, EGFR-10001 inhibited the growth of ovarian carcinoma (A2780 ADR) in mice. The tumor volume was reduced in the 3D model (see Figures 5A to 5C).

[0355] Uterine sarcoma MES-SA DX5 cells were transplanted into nude mice to model uterine sarcoma. Anti-ABCB1 antibody B1 is effective in the MES-SADX5 uterine sarcoma model in nude mice. B1.261, B1.129, and B1.223 were used as single agents and in combination with paclitaxel in mice. It reduced tumor volume in a uterine sarcoma model (MES-SADX5) (see Figure 6).

[0356] Figure 5C. KPA02 is an anti-ABCB1 antibody known as MRK16. KPA08 is an anti-ABCB1 antibody known as MRK16 and 15D3. This anti-ABCB1 antibody was generated by combining two anti-ABCB1 antibodies: KPA08 The antibody contains two heavy chains derived from 15D3 and two light chains derived from MRK16. KPA01 is known as 15D3. KPA06 is an anti-ABCB1 antibody. KPA06 is an anti-CD47 antibody 5F9.

[0357] The 15D3 antibody (KPA01) did not sensitize MES-SA DX5 uterine sarcoma to paclitaxel. The 1.129 antibody has been shown to inhibit tumor growth both as a single agent and in combination with paclitaxel. Both B1.261 and B.223 were more effective than the 15D3 antibody (KPA01) in inhibiting the MES-SADX5 molecule. Miyazaki sarcoma was sensitized to paclitaxel. See Figure 6.

[0358] MES-SA DX5 cells were transplanted into NSG mice to model uterine sarcoma. The antibody is efficacious in the MES-SADX5 uterine sarcoma model in NSG mice. -SA DX5 uterine sarcoma sensitizes to paclitaxel.

[0359] Figure 8 shows that ABCB1 expression is required for resistance of MES-SADX5 uterine sarcoma to paclitaxel. MES-SADX5 cells lacking ABCB1 expression (DX5-B1 KO) were used to generate MES-SADX5 Uterine sarcoma is sensitive to paclitaxel.

[0360] Example 3: Epitope mapping of anti-MDR1 antibodies The binding of the anti-MDR1 mAbs listed in Table 2 to the two major extracellular loops 1 and 4 of MDR1 was determined. To achieve this, various mutations were constructed to alter or replace these two loops. To examine binding to the cynomolgus extracellular loops, standard techniques were used to assay the cynomolgus extracellular loops. Replacement of extracellular loop 1 and / or loop 4 of MDR1 with the human loops in human MDR1 The binding dependency on the normal human loop was examined. To address this issue, we also generated MDR1 variants with deletions 79–102 and 82–99 in loop 1. Loop 1 is also called ECD1. Loop 4 is also called ECD4. These mutant MDR1 efflux Pump variants were expressed on 293T cells using standard methods and expressed in various anti-MDR1 mAbs. We generated cells that can be used to distinguish between different binding sites of different anti-MD antibodies. The cells were incubated with R1 mAb for approximately 1 hour at 4°C. The cells were then resuspended in FACS buffer (containing 0.5% BSA). The samples were washed twice with PBS. Alexa647-labeled goat anti-human antibody was added as a secondary antibody, and the samples were stored on ice. The samples were washed again and analyzed by flow cytometry. analyzed.

[0361] Figure 9 summarizes findings from preliminary epitope mapping with mutant human MDR1. "+" indicates that the listed ECD was required for antibody binding (WT human ABCB1 (Based on a significant decrease in binding to mutant ABCB1 lacking the ECD compared to the wild-type ABCB1.) This indicates that the ECD was not required for antibody binding (compared to WT human ABCB1 lacking the ECD). (Based on the lack of significant changes in binding to mutant ABCB1). For example, anti-MDR1 mAb B1.1 29 requires ECD1 and ECD4 to bind to human MDR1. All anti-MDR1 antibodies The KPA01 and KPA08 antibodies do not require ECD1 for binding to ABCB1. The data show that many of the anti-B1 antibodies listed in Figure 9 are "+" for binding both ECD1 and ECD4. As shown in Figure 1, the antibody binds to an epitope containing a sequence present in ECD1 and ECD4. In contrast, the KPA01 and KPA08 antibodies target epitopes containing the ECD4 sequence but not the ECD1 sequence. ECD1 refers to the first extracellular domain of human ABCB1, and ECD4 refers to the first extracellular domain of human ABCB1. "Other" refers to the remaining extracellular domains 2-3 and 4 of human ABCB1. and 5-6. Only binding to ECD1 and ECD4 was tested.

[0362] Example 4: Characterization of anti-MDR1 antibodies Anti-MDR1 mAb B1.129 and its humanized version were injected into 293T cells overexpressing human MDR1. Binding to 293T cells transfected with either the cynomolgus MDR1 or cynomolgus MDR1 was assessed. Figures 10A-10B show that B1.129 mAb inhibits human (hB1, Figure 10A) and cynomolgus monkey (cB1, Figure 10B) MD. B1.129.hz1 binds to both hB1 and cB1 with lower affinity. However, B1.129.hz2 did not show significant binding to either hB1 or cB1. The B1.129.hz2 antibody is a humanized version of the B1.129.huH1-huL1 (KV3) antibody described in Table 2. ) and B1.129.huH1-huL1(KV1). Figure 10C summarizes the binding data for the B1.129 antibody. To promise.

[0363] Anti-MDR1 mAb B1.28 and its humanized version were administered to the 293T cell line, which overexpresses human MDR1. Alternatively, binding to 293T cells transfected with cynomolgus monkey MDR1 was assessed. Figure 11 shows that B1.28 mAb binds to both hB1 and cB1. B1.28 hz1 mAb binds to B1 The B1.28.hz1 antibody bound to both hB1 and cB1 with lower affinity compared to the .28 mAb. It is the same as the humanized version B1.28 human1 described in

[0364] Figure 12 shows the binding of anti-MDR1 mAb B1.261 and its two humanized versions hB1 and cB1. Combine with.

[0365] Responds to the efflux of MDR1 substrates calcein, DIOC2, and rhodamine The effect of anti-MDR1 antibodies on the efflux blockade was assessed as described in the efflux blockade experimental procedure. The data is summarized in Figure 13.

[0366] As can be seen in Figure 13, B1.129 mAb inhibited calcitonin to a greater extent than anti-MDR1 antibody 15D3. The B1.129 mAb also inhibited DIOC2 efflux in two different hB1-expressing cell lines. In contrast, 15D3 inhibited DIOC2 efflux in both cell lines. showed no observable effect on rhodamine efflux in the 293T.hB1 cell line (-) and showed a low inhibition of rhodamine efflux in the 293T.hB1 cell line (-). (+ / -) but had no effect on rhodamine efflux in MES-SA / DX5 cells. .

[0367] Example 5: Chemosensitization of cancer cells by anti-MDR1 antibodies The anti-MDR1 antibodies listed in Table 2 were tested against vincristine-treated N6ADR cells. The effects of many anti-MDR1 mAbs on biotoxicity were tested. See Figures 4A-4D. Table 7 shows that many anti-MDR1 mAbs The cytotoxicity of cristine was investigated using another cytotoxic agent, Valspodar. This shows that the results have increased to levels comparable to or even exceeding those achieved by the Many of the anti-MDR1 antibodies shown in 2 potentiated the chemotoxicity of vincristine by more than 40-fold. These antibodies are shown in italics in Table 7. JPEG2026041756000030.jpg217164JPEG2026041756000031.jpg70158

[0368] Example 6: Generation of bispecific antibodies that bind to MDR1 and CD47 15D3VH:B1-28VL:: B1-28VL:5F9VH bispecific antibody The variable heavy chain (VH) of the anti-MDR1 antibody 15D3, the variable heavy chain of the anti-CD47 antibody 5F9, and the variable heavy chain of the anti-MDR1 antibody "B1- A bispecific antibody was produced with a common variable light chain from each of the 15D3 and 15D3 VH chains (see Table 2). A humanized version was generated, which contains framework regions from the human IGHV3 group. These variable heavy chains (HC) are referred to herein as 15D3. Hz0 (humanized 15D3 HC); 15D3 Hz1 (humanized 15D3 HC + N56Q substitution in CDR H2); and It is called 15D3 Hz2 (humanized 15D3 HC + N56S substitution in CDR H2) and has the following amino acid sequence: do. 15D3 Hz0: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:350) 15D3 Hz1: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:351) 15D3 Hz2: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:352)

[0369] The sequence of the 5F9 antibody variable heavy chain is as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:294) The sequence of the humanized B1-28 VL chain is as follows: DVVLTQSPLSLPVTLGQPASISCRSSQNIVHSTGNTYLDWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYFCFQGSHIPRTFGQGTKLEIK (SEQ ID NO:20) The sequence of the humanized B1-28 VH chain is as follows: EVQLVESGGGLVKPGGSLRLSCAASGFTFGLYTMSWVRQAPGKGLEWVATISSGGSNTYYPDSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCARYYRYDAWFAYWGQGTLVTVSS (SEQ ID NO:19)

[0370] The bispecific antibodies contained either the 15D3Hz1 or 15D3Hz2 variable regions. The heavy chain region was fused to a human IgG1 Fc region with charge pair substitutions K392D and K409D, and the 5F9 variable The heavy chain region is fused to a human IgG1 Fc region with the charge pair substitutions E356K and D399K.

[0371] The bispecific antibodies were characterized by binding titration and chemical toxicity assays.

[0372] Cell binding assay Antibody binding to cells was assessed by flow cytometry. 293T cells stably transfected with ABCB1 (293T_ABCB1_OX) were analyzed by flow cytometry. -Wash once in buffer (PBS + 2% FBS + 0.02% sodium azide) and analyze by flow cytometry Resuspend cells at 2 x 10^6 cells / mL in buffer and place in a 96-well microtiter plate at 0.1 mL / well. Recombinant antibodies were added to cells at 5 μg / mL for initial binding confirmation. or serially diluted from 100 μg / mL in flow cytometry buffer. After incubation for 30 minutes, the cells were washed twice with flow cytometry buffer. The antibodies were detected with PE-labeled F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch), and the Att The antibody concentrations that gave half the maximal response were evaluated using a NxT flow cytometer. EC50 is the antibody concentration that gave half the maximal response. It is calculated as:

[0373] Cytotoxicity assay The effect of the antibody on vincristine cytotoxicity was investigated using N6 / ADR (human acute lymphoblastic leukemia) The ALL (Allergy) cell line NALM6 (doxorubicin-selected, B1-positive variant) was evaluated. In a white flat-bottom 96-well tissue culture plate, add 0.05 mL of assay medium (RPMI-1640 + 10% Cells were plated at 5000 cells / well in 100 μg / mL (2× FBS). Vincristine was added at 100 μg / mL (2× FBS). 7 μM (2× final concentration) of test or control antibody or 7 μM (2× final concentration) of a small molecule B1 inhibitor Serial dilutions from 200 μM in assay medium containing ruspodar yielded 2× final assay Equal volumes (0.05 mL) of the vincristine / antibody mixture were added to a 96-well plate. The cells were added to N6 / ADR cells in a bronchodilator. The plates were then incubated at 37°C in 5% CO2. After 72 hours, the plates were equilibrated to room temperature and incubated at 4°C for 1 hour at RT. Cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. Luminescence was measured using a Molecular Devices® FlexStation® 3 Multimode The data were analyzed using a microplate reader and analyzed using GraphPad Prism 8.0 software. The half-maximal inhibitory concentration (IC50) was the concentration of a drug ( vincristine or other chemotherapy cytotoxic agent).

[0374] The cell binding of bispecific antibodies (BisAb-1 and BisAb-2) to 293T_ABCB1_OX cells is summarized in Table 3. To promise.

[0375] [Table 3]

[0376] KT14 refers to CD47. The KT14 antibody (Ab) in Table 3 is the anti-CD47 antibody 5F9 (amino acid sequence: (See Table 5 for details.) In both BisAb-1 and BisAb-2, the HC from the anti-CD47 antibody 5F9 was used. It was.

[0377] The effect of antibodies on vincristine cytotoxicity is summarized in Table 4. hIgG1, 15D3 and MR The K16 antibody did not enhance vincristine-mediated cytotoxicity, whereas the Bis-Ab-1 and BisAb-2 antibodies increased the cytotoxicity of vincristine.

[0378] [Table 4]

[0379] [Table 5] JPEG2026041756000035.jpg226156JPEG2026041756000036.jpg214156JPEG2026041756000037.jpg22715 8JPEG2026041756000038.jpg221157JPEG2026041756000039.jpg227156JPEG2026041756000040.jpg88160

[0380] Table 6 lists the nomenclature used for the antibodies used to generate the data in the figures. To summarize:

[0381] [Table 6]

[0382] The heavy chain of the anti-MDR1 antibody 15D3 was combined with the heavy chain of the anti-CD47 antibody 5F9 and a different light chain from an anti-MDR1 antibody. The binding of these bispecific antibodies to C6 cells overexpressing human B1 was investigated. The data is shown in Figure 14.

[0383] Rat fibroblast C6 cells were transfected with stable expression of hABCB1 or hKT14. We generated cells expressing 5F9 and anti-ABCB1 antibodies in mouse KT14 and mouse KT14 cell lines, respectively. Since the C6 cell line has little affinity for ABCB1, the C6 cell line was used to investigate the expression of the C6 The affinity of KT14 / Fc / KT14 Ab to the human target was measured. le Arm) format, i.e., one heavy chain and one light chain only.

[0384] Figure 15 shows the effect of C6 cells overexpressing cynomolgus monkey CD47 (cKT14) or human CD47 (hKT14). The binding of these bispecific antibodies to the flow Recombinant proteins were assessed by biolayer interferometry (BLI) using cytometry. Octet KD- was recombinantly analyzed by BioLayer Interferometry (BLI) using the ForteBio Octet. The antibody binding to human CD47 (rhCD47) was measured. After loading, the cells were exposed to rhCD47 at 300 nM, 100 nM, and 33 nM. Binding constants were calculated for the cleavage and dissociation. A 1:1 curve fit was applied, and a global fit was used. BsAb binding appears to require a large amount of B1, whereas CD47 binding is required for Increasing the amount of B1 does not increase the binding affinity. Therefore, BsAb is specific to cells overexpressing B1. Since CD47 is expressed on many normal cells, low affinity for CD47 is expected. Compatibility is advantageous in reducing off-target effects.

[0385] B1+ / CD47+ MES-SA-DX5 cells (hereafter referred to as DX5WT cells), B1 gene derived from DX5WT cells knockout and CD47 gene knockout cell lines (hereafter referred to as DX5 B1 KO (B1- / CD4 7+) and DX5 CD47 KO (B1+ / CD47-) cells) were used to perform double immunohistochemistry using anti-MDR1 and anti-CD47 antibodies. The binding selectivity of the specific antibodies was evaluated.

[0386] Prior to the addition of antibodies, DX5 WT cells and DX5 CD47 KO cells were cultured using CellTrace TM by CellTrace and CellTrace TM Stained with Far Red (Life Technologies, NY) and DX5 B1 K 0 cells were left unstained. Cells were cultured at 6 × 10 6 cells / ml (2×10 6 cells / ml of each cell population) The final concentration was 3 x 10 5 Serially dilute cells / well The cells were then incubated with the selected antibodies at room temperature for 1 hour. Then, the cells were washed three times with FACS buffer. The cell-binding antibody was a PE-conjugated goat anti-human IgG Fcγ-specific antibody (Jackson ImmunoResearch). It was detected using the search.

[0387] Flow cytometry was performed using Attune NXT (Thermo Fisher Scientific) and tracer Gating of individual cell populations based on dye expression or exclusion and physical scattering properties Doublets were excluded by centrifugation. Antibody binding was measured by mean fluorescence intensity (MFI).

[0388] Figure 16 shows the results of the listed bispecific antibodies against DX5 WT, DX5 B1 KO, and DX5 CD47 KO cell lines. Binding selectivity titration of

[0389] Figures 17A and 17B show human and cynomolgus MDR1-expressing cells after humanization of HC and LC. Figure 1 shows the binding of anti-MDR1 and anti-CD47 bispecific antibodies to humanized HC and LC. The acid sequence is as follows: 15D3 hmz DD IgG1 (HC): EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:350) B1.28.huL1 (LC): DVVLTQSPLSLPVTLGQPASISCRSSQNIVHSTGNTYLDWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYFCFQGSHIPRTFGQGTKLEIK (SEQ ID NO:20) B1.28.huL2 (LC): DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO:21) B1.28.huL3 (LC): EVVLTQSPATLSLSPGERATLSCRSSQNIVHSTGNTYLDWYQQKPGQSPRLLIYKVSNRFSGVPARFSGSGSGTDFTLTI SSLEPEDFAVYYCFQGSHIPRTFGGGTKLEIK (SEQ ID NO:22) B1V6.CDRv1.hmzLC: DIVMTQTPLSLPVTLGDPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO:367) B1V6.CDRv2.hmzLC: DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO:311) B1V6.CDRv3.hmzLC: DIVLTQTPLSLPVSLGDPASISCRSSQSLVHSSGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO:368)

[0390] To find the optimal bispecific antibody, many of the heavy chains of the anti-MDR1 antibodies listed in Table 2 were used with anti-C D47 antibody 5F9 heavy chain and different light chains from anti-MDR1 antibodies. The anti-MDR1 antibodies listed in Table 2 were derived.

[0391] for CD47 using ELISA or CD47-expressing cells, and for ABCB1-expressing cells. The results of assaying the binding of various bispecific antibodies by the authors are shown in Figures 18A and 18B.

[0392] KPB1 VL / CDR3 LC was generated from the KJB1-6 light chain antibody by introducing mutations into VH CDR3.

[0393] KPB1-6 LC sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK (SEQ ID NO:369) VL CDR1: RSSQSLVHSNGNTYLE (SEQ ID NO:290) VL CDR2: KVSNRFS (SEQ ID NO:12) VL CDR3: QGSHVPFT (SEQ ID NO:370) KPB1 VL / CDR3 LC sequence: DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO:311) VL CDR1: RSSQSLVHSNGNTYLE (SEQ ID NO:290) VL CDR2: KVSNRFS (SEQ ID NO:12) VL CDR3: QGSHFPRT (SEQ ID NO:348)

[0394] Unless otherwise specified, all of the bispecific antibodies disclosed herein are directed to anti-MDR1 antibodies. HCs derived from anti-tumor-associated antigen antibodies (e.g., anti-CD47 antibodies), and anti-MDR1 antibodies These antibodies are listed in the format VH / VH / VL or VH:VL::VL:VH. It will be displayed.

[0395] Example 7: Generation of bispecific antibodies with non-traditional HC and LC configurations The monospecific and bispecific antibodies disclosed herein can be synthesized using the methods shown in FIG. The heavy and light chains in the mat may be included.

[0396] Anti-MDR1 and anti-CD47 bispecific antibodies were synthesized in scFv-Fab-Fc format as shown in Figure 20. The Fc region of the anti-CD47 arm of the bispecific antibody was created by amino acid substitution. The Fc region of the anti-MDR-1 arm is shown as having an amino acid substitution However, the anti-CD47 arm does not contain a knob. The knob and hole mutations may be reversed so that the anti-MDR1 arm contains the hole. Heterologous antibodies were produced. KT14.5F9hu scFv-B1.28.hu13 KT14.B6H12hu scFv-B1.28.hu13 KT14.5F9hu scFv-B1.261.hu1 KT14.B6H12hu scFv-B1.261.hu1

[0397] KT14.5F9hu scFv and KT14.B6H12hu scFv are human IgG1 Fc domains with "knob" mutations. The Fc region contains a "hole" mutation that facilitates pairing with the Fc region of an anti-MDR1 antibody containing the "hole" mutation. KT14.5F9hu scFv-hIgG1_knob has the following amino acid sequence: JPEG2026041756000042.jpg62162HCDR 1-3 are shown in bold. LCDR 1-3 are shown in bold and underlined.

[0398] KT14.B6H12hu scFv-hIgG1_knob has the following amino acid sequence: JPEG2026041756000043.jpg61162HCDR 1-3 are shown in bold. LCDR 1-3 are shown in bold and underlined.

[0399] B1.28.hu13-hIgG1_hole has the following amino acid sequence: JPEG2026041756000044.jpg55162HCDR 1 to 3 are shown in bold.

[0400] The B1.28.LC used has the following amino acid sequence: JPEG2026041756000045.jpg29161LCDR 1 to 3 are displayed in bold and underlined.

[0401] B1.261.huH1.hIgG1_hole has the following amino acid sequence: JPEG2026041756000046.jpg55161HCDR 1 to 3 are shown in bold.

[0402] B1.261.huL1 has the following amino acid sequence: JPEG2026041756000047.jpg28161LCDR 1 to 3 are displayed in bold and underlined.

[0403] Figure 21 shows that the KT14.5F9hu scFv-B1.28.hu13 and KT14.B6H12hu scFv-B1.28.hu13 antibodies inhibited C6 It is shown to bind to both human CD47 and human MDR1 expressed on the surface of cells.

[0404] Figure 22 shows that the KT14.5F9hu scFv-B1.261.hu1 and KT14.B6H12hu scFv-B1.261.hu1 antibodies inhibited C6 It is shown to bind to both human CD47 and human MDR1 expressed on the surface of cells.

[0405] Example 8: In vivo efficacy of anti-MDR1 and anti-CD47 antibodies Figures 23A-23C show the efficacy of anti-MDR1 and anti-MDR1 inhibitors in reducing tumor volume in the MES-SA-DX5 model. Figure 23A shows the in vivo efficacy of B1-188 VH:MRK16LC::MRK1 and anti-CD47 bispecific antibodies. 6LC:5F9 HC antibody (B1-188 / KT14 / MRK16) and B1-225 VH:MRK16 LC::MRK16 LC:5F9 HC antibody (B1-225 / KT14 / MRK16) reduced tumor volume alone and in combination with paclitaxel treatment B1-225 / KT14 / MRK16 is a 15D3 VH:MRK16LC::MRK16LC:5F9 HC antibody (15D3 / KT14 / MRK16) have similar efficacy to KBisP1.1 antibodies.

[0406] Figure 23B shows that each of the bispecific antibodies was tested as a single agent and in combination with paclitaxel. and tumor growth to levels lower than those observed with paclitaxel and negative control antibodies. Indicates a decrease in volume.

[0407] Figure 23C shows the results of replacing MRK16 LC with LC from anti-MDR1 antibodies B1.VL6, B1.225v1, or B1.27. This has demonstrated the efficacy of the bispecific antibody as a single agent or in combination with paclitaxel. Indicates that it is enhanced.

[0408] Example 9: Generation of bispecific antibodies that bind to MDR-1 and Her2 The heavy chains of the anti-MDR1 antibodies shown in Table 2 were fused to the anti-Her2 antibodies trastuzumab or pertuzumab. Combined with the heavy chain of Br.

[0409] Trastuzumab light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP EDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:376) Trastuzumab heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:320) Pertuzumab heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLY LQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:324) Pertuzumab light chain sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:377)

[0410] The designation "KT-1" is used herein to refer to the trastuzumab antibody. The designation "KT-1-2" is used herein to refer to the pertuzumab antibody. Figure 24 shows the use of a bispecific antibody format to identify combinations that bind to both antigens. The following lists the various combinations of anti-MDR1 heavy chains, anti-HER2 heavy chains, and anti-MDR1 common light chains that have been tested in this study. Binding to HER2 was determined using the HER2-expressin...

Claims

1. An antibody that specifically binds to multidrug resistance protein 1 (MDR1), wherein the antibody is listed in Table 2. The heavy chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) of the pair of variable heavy chain (VH) and variable light chain (VL) regions of competes for binding to MDR1 with an antibody comprising light chain CDRs 1-3 (LCDRs 1-3); antibody.

2. An antibody comprising a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2, and M The antibody of claim 1, which competes for binding to DR1.

3. Selected from the group consisting of antibodies B1.28, B1.261, B1.129, B1.225, and B1.223 listed in Table 2 The antibody of claim 1, which competes for binding to MDR1 with the selected antibody.

4. The antibody of claim 1, comprising the HCDR of the VH region of an antibody listed in Table 2.

5. The antibody of claim 1 or 2, comprising the LCDR of the VL region of an antibody listed in Table 2.

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

7. An antibody molecule that specifically binds to multidrug resistance protein 1 (MDR1), The antigen-binding site is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 sex-determining regions 1–3 (HCDRs 1–3) and light chain CDRs 1–3 (LCDRs 1–3); HCDRs 1-3 of the VH region of an antibody listed in Table 2; or LCDRs 1 to 3 of the VL regions of the antibodies listed in Table 2 An antibody molecule comprising:

8. comprising HCDRs 1-3 and LCDRs 1-3 of a pair of VH and VL regions of an antibody listed in Table 2; The antibody molecule of claim 7.

9. The antibody of claim 7, comprising HCDRs 1 to 3 of the VH region of a first antibody listed in Table 2.

10. The antibody molecule of claim 7, comprising LCDRs 1 to 3 of the VL region of a second antibody in Table 2.

11. Claims comprising the variable light (VL) chain and / or variable heavy (VH) chain of an antibody listed in Table 2.

7. An antibody molecule according to claim 7.

12. (i) HCDRs 1 to 3 of the VH region of the B1.28 antibody and LCDRs 1 to 3 of the VL region of the B1.28 antibody; (ii) HCDRs 1 to 3 of the VH region of the B1.261 antibody and LCDRs 1 to 3 of the VL region of the B1.261 antibody; (iii) HCDRs 1 to 3 of the VH region of the B1.129 antibody and LCDRs 1 to 3 of the VL region of the B1.129 antibody; (iv) HCDRs 1 to 3 of the VH region of the B1.225 antibody and LCDRs 1 to 3 of the VL region of the B1.225 antibody; (v) HCDRs 1 to 3 of the VH region of the B1.188 antibody and LCDRs 1 to 3 of the VL region of the B1.188 antibody; (vi) HCDRs 1 to 3 of the VH region of the B1.27 antibody and LCDRs 1 to 3 of the VL region of the B1.27 antibody; or (vii) HCDRs 1 to 3 of the VH region of the B1.223 antibody and LCDRs 1 to 3 of the VL region of the B1.223 antibody wherein the VH and VL regions are as set forth in Table 2. The antibody molecule of claim 8

13. The antibody according to any one of claims 7 to 12, which binds to human MDR1 and cynomolgus monkey MDR1. antibody molecule.

14. HCDRs 1 to 3 of the VH region of the B1.129 antibody and LCDRs 1 to 3 of the VL region of the B1.129 antibody, and V comprising HCDRs 1-3 of the H region and LCDRs 1-3 of the VL region of the B1.28 antibody, or The antibody of claim 13, comprising HCDRs 1 to 3 of the VH region and LCDRs 1 to 3 of the VL region of the B1.261 antibody. body molecules.

15. A compound according to any one of claims 7 to 14, which binds to loop 1 in the extracellular domain of MDR1. The antibody molecule described.

16. HCDRs 1 to 3 of the VH region and LCDRs 1 to 3 of the VL region of the B1.129 antibody, or the VH region of the B1.223 antibody The antibody molecule of claim 15, comprising HCDRs 1 to 3 of the VL region and LCDRs 1 to 3 of the VL region.

17. Calcein, daunorubicin (Dioc2), and rhodamine by cancer cells The antibody molecule of any one of claims 7 to 16, which inhibits the above-mentioned excretion.

18. The antibody molecule of claim 17, wherein the cancer cells are 293T cells that overexpress MDR1.

19. The antibody molecule of claim 17, wherein the cancer cells are MES-SA-DX5 cancer cells.

20. 21. The method of claim 17, comprising HCDRs 1 to 3 of the VH region and LCDRs 1 to 3 of the VL region of the B1.129 antibody. The antibody molecule of any one of claims 1 to 4.

21. An antibody molecule that specifically binds to MDR1 expressed on a cell surface, comprising any of the following: An antibody molecule comprising: (i) a heavy chain complementarity-determining region (HCDR) 1 comprising the amino acid sequence GFTFSRY (SEQ ID NO: 343); HCDR2 containing the amino acid sequence SSGGGN (SEQ ID NO: 344); and a variable heavy (VH) chain comprising an HCDR3 comprising the amino acid sequence RSSQNIVHSTGNTYLD (sequence number light chain complementarity determining region (LCDR) 1 containing the amino acid sequence KVSNRFS (SEQ ID NO: 12); and an LCDR3 comprising the amino acid sequence FQGSHIPRT (SEQ ID NO: 13). L) chain; (ii) Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 287) a VH chain comprising the amino acid sequence DVVLTQSPLSLPVTLGQPASISCRSSQNIVHSTGNTYLDWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYFCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 20) a VL chain comprising: (iii) Amino acid sequence EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVL DSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 309) a heavy chain comprising the amino acid sequence DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 21) a light chain comprising: (iv) HCDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO: 346); and and an HCDR2 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308), a VH chain; and an LCDR1 comprising the amino acid sequence RSSQNIVHSTGNTYLD (SEQ ID NO: 11); LCDR2 containing the sequence KVSNRFS (SEQ ID NO: 12); and LCDR3 containing the amino acid sequence FQGSHIPRT (SEQ ID NO: 13). a VL chain comprising an LCDR3 comprising: (v) Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) VH chain and amino acid sequence comprising DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 21) a light chain comprising: (vi) Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 310) heavy chain and amino acid sequence comprising DVVLTQSPLSLPVTPGEPASISCRSSQNIVHSTGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCFQGSHIPRTFGQGTKLEIK (SEQ ID NO: 21) a light chain comprising: (vii) an HCDR1 comprising the amino acid sequence GFTFSRY (SEQ ID NO: 343); and and HCDR2 comprising the amino acid sequence GAGDAWFAY (SEQ ID NO: 345). a VH chain comprising the amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 290); and an LCDR1 comprising the amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 290); LCDR2 containing the amino acid sequence KVSNRFS (SEQ ID NO: 12); and LCDR3 containing the amino acid sequence QGSHFPRT (SEQ ID NO: 348). and an LCDR3 comprising: (viii) Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 287) and a VH chain comprising the amino acid sequence DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) a VL chain comprising: (ix) Amino acid sequence EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVL DSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 309) heavy chain and amino acid sequence comprising DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) a light chain comprising: (x) HCDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO: 346); and and an HCDR2 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308), a VH chain; and an LCDR1 comprising the amino acid sequence RSSQSLVHSNGNTYLE (SEQ ID NO: 290); LCDR2 containing the sequence KVSNRFS (SEQ ID NO: 12); and LCDR3 containing the amino acid sequence QGSHFPRT (SEQ ID NO: 348). a VL chain comprising an LCDR3 comprising: (xi) Amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY VH chain comprising the amino acid sequence MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) a light chain comprising: (xii) amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 310) heavy chain and amino acid sequence comprising DIVMTQSPLSLPVSLGDPASISCRSSQSLVHSNGNTYLEYYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 311) a light chain comprising: (xiii) HCDR1 comprising the amino acid sequence GFTFSRY (SEQ ID NO: 343); and and HCDR2 comprising the amino acid sequence GAGDAWFAY (SEQ ID NO: 345). a VH chain comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 39); and an LCDR1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 39); LCDR2 containing the amino acid sequence KVSKRFS (SEQ ID NO: 40); and LCDR3 containing the amino acid sequence FQASHFPRT (SEQ ID NO: 329). and an LCDR3 comprising: (xiv) Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 287) and a VH chain comprising the amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) a VL chain comprising: (xv) amino acid sequence EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLY LQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVL DSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 309) heavy chain and amino acid sequence comprising DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) a VL chain comprising: (xvi) HCDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO: 346); and and HCDR2 comprising the amino acid sequence GGYRAMDY (SEQ ID NO: 308). , a VH chain; and an LCDR1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 39); LCDR2 containing the amino acid sequence KVSKRFS (SEQ ID NO: 40); and LCDR3 containing the amino acid sequence FQASHFPRT (SEQ ID NO: 329). a VL chain comprising an LCDR3; (xvii) amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) VH chain and amino acid sequence comprising DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) a light chain comprising: (xviii) amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 310) heavy chain and amino acid sequence comprising DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 313) A light chain comprising:

22. A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and a tumor-associated antigen (TAA) and comprising two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain. wherein each of the VL chains comprises an antigen-binding site for MDR1, and the first VH chain comprises an antigen-binding site for MDR 1, and the second VH chain comprises an antigen-binding site for TAA. wherein said second VH chain binds to a TAA when paired with one of said light chains. 。

23. The antigen-binding sites of the two VL chains correspond to light chain CDRs 1-3 (LCDRs 1-3) of the antibodies listed in Table 2.

23. The bispecific antibody molecule of claim 22, comprising:

24. the antigen-binding site of the first VH chain comprises heavy chain CDRs 1-3 (HCDRs 1-3) of an antibody listed in Table 2 24. The bispecific antibody molecule of claim 22 or 23, comprising:

25. the first VH chain comprises HCDRs 1-3 of an anti-MDR1 antibody other than an antibody listed in Table 2, and optionally wherein the first VH chain comprises the amino acid sequence of the VH chain of an anti-MDR1 antibody other than the antibodies listed in Table 2.

24. A bispecific antibody molecule according to claim 22 or 23.

26. 26. The bispecific antibody molecule of claim 25, wherein the anti-MDR1 antibody comprises: Amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO: 293) the VH chain of the 15D3 antibody having the formula: Amino acid sequence AVQLQQSGPELVKTGASVKISCKASGYSFSNYYIHWVKQSHGKSLEWIGFISCYNGATFYNQKFKGKATFTVDTSSSTAY MKFNSLTFEDSAVYYCARLPIQFGNFYPMDYWGQGTSVTVSS (SEQ ID NO: 288) or a VH chain of a UIC2 antibody having the formula: Amino acid sequence EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLY LQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA (SEQ ID NO: 286) The VH chain of the MRK16 antibody having the following structure:

27. the anti-MDR1 antibody has the amino acid sequence EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 292) wherein X comprises a VH chain having 2 is N, Q or S, and optionally X 2 is Q or S, claim 27. The bispecific antibody molecule of claim 26.

28. the VL chain comprises LCDRs 1 to 3 of the B1-28 antibody as set forth in Table 2; Optionally, the VL chain comprises the amino acid sequence of the VL chain of the B1-28 antibody listed in Table 2; or 、 The VL chain has the amino acid sequence LWVPGSTGDVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS Anti-MDR1 having a VL chain comprising GTDFTLKISRVEAEDLGVYYCFQGSHFPRTFGGGTKLEIK (SEQ ID NO: 289) comprising LCDRs 1 to 3 of the V6 antibody; Optionally, the VL chain comprises the amino acid sequence of the VL chain of the V6 antibody. A bispecific antibody molecule according to any one of claims 22 to 27.

29. the VL chain comprises LCDRs 1-3 of an anti-MDR1 antibody other than an antibody listed in Table 2, and optionally the light chain comprises:

25. The antibody of claim 22 or 24, comprising the amino acid sequence of the VL chain of an antibody other than an antibody listed in Table 2.

2. A bispecific antibody molecule as described herein.

30. the anti-MDR1 antibody Amino acid sequence DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 283) the variable light chain of the MRK16 antibody comprising: Amino acid sequence DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 284) the variable light chain of the 15D3 antibody comprising: Amino acid sequence DVVMTQTPRSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHIPPWTFGGGTKLDIK (SEQ ID NO: 285) The variable light chain of a UIC2 antibody comprising 30. The bispecific antibody molecule of claim 29, comprising LCDRs 1 to 3 of:

31. the antibody inhibits MDR1-mediated efflux when bound to cells expressing MDR1; An antibody molecule or bispecific antibody molecule according to any one of claims 1 to 4.

32. the antibody is capable of increasing the sensitivity of cancer cells to treatment with a chemotherapeutic agent; wherein the half maximal inhibitory concentration (IC50) of the chemotherapeutic agent when co-administered with the antibody is: The following array EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLY LQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO: 293) and a VH chain having The following array DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 284) VL chain having at least 5-fold lower than the IC50 of the chemotherapeutic agent when co-administered with an anti-MDR1 antibody comprising A bispecific antibody molecule according to any one of claims 22 to 31.

33. The bispecific antibody molecule of any one of claims 22 to 32, wherein the TAA is CD47. 。

34. The bispecific antibody comprises HCDRs 1 to 3 of the VH region of the anti-CD47 antibody 5F9, wherein the VH region comprises: The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) or The bispecific antibody comprises HCDRs 1 to 3 of the VH region of the anti-CD47 antibody B6H12, and the VH region is The amino acid sequence below EVQLVESGGDLVKPGGSLKLSCAASGTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYYPDSVKGRFTISRDNAKNTLY LQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSS (SEQ ID NO: 295) 34. The bispecific antibody molecule of claim 33, comprising:

35. the first VH chain comprises HCDRs 1-3 of a VH chain derived from an anti-MDR1 antibody having the following HCDR sequences: HCDR1: RYTMS (SEQ ID NO: 301), HCDR2: TISSGGGNTYYPDSVKG (SEQ ID NO: 302), TISSGGGQTY YPDSVKG (SEQ ID NO: 303), or TISSGGGSTYYPDSVKG (SEQ ID NO: 304), and HCDR3: ARY GAGDAWFAY (SEQ ID NO: 349); the VL chain comprises LCDRs 1 to 3 of the VL region of the B1-28 antibody, respectively, as set forth in Table 2, The R sequences are LCDR1: RSSQNIVHSTGNTYLD (SEQ ID NO: 11), LCDR2: KVSNRFS (SEQ ID NO: 12), and and LCDR3: FQGSHIPRT (SEQ ID NO: 13), 35. The bispecific antibody molecule of claim 34.

36. the first VH chain EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX2TYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 291) wherein X2 is N, Q, or S; the second VH chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAY MELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 294) and an amino acid sequence comprising: The two VL chains comprise amino acid sequences comprising the VL region chains of the B1-28 antibody set forth in Table 2.

36. The bispecific antibody molecule of claim 35.

37. The first VH chain is selected from the group consisting of (i) B1.28 antibody; (ii) B1.30 antibody; (iii) B1.89 antibody; and (i) (v) HCDRs 1 to 3 of the VH chain of the B1.129 antibody; (v) B1.225; (vi) B1.261 antibody; or 15D3 or comprises HCDRs 1-3 of the VH chain of the MRK16 antibody; The identical VL chains each comprise: (i) LCDRs 1 to 3 of the VL chain of the MRK16 antibody; or (ii) LCDRs 1 to 3 of the VL chain of the V6 antibody. comprising LCDRs 1-3 of the VL chain; and the second VH chain comprises HCDRs 1 to 3 of the VH chain of the 5F9 antibody or the B6H12 antibody; 23. The bispecific antibody molecule of claim 22.

38. The first VH chain is selected from the group consisting of (i) B1.188 antibody; (ii) B1.28 antibody; (iii) B1.261 antibody, as listed in Table 2; (iv) the VH chain of the B1.129 antibody; or (v) the VL chain of the B1.223 antibody, each comprising LCDRs 1 to 3 of the VL chain of the MRK16 antibody; and the second VH chain comprises LCDRs 1 to 3 of the VL chain of the 5F9 antibody or B6H12 23. The bispecific antibody molecule of claim 22, comprising HCDRs 1 to 3 of the VH chain of the antibody.

39. The first VH chain is selected from the group consisting of (i) B1.188 antibody; (ii) B1.28 antibody; (iii) B1.261 antibody, as listed in Table 2; (iv) the VH chain of the B1.129 antibody; or (v) the VL chain of the B1.223 antibody, each of which is an MRK1 6 antibody, and the second VH chain comprises the VH chain of the 5F9 antibody or the B6H12 antibody.

9. The bispecific antibody molecule according to claim 8.

40. the first VH chain comprises HCDRs 1 to 3 of the VH chain of the B1.225 antibody listed in Table 2, and the same VL chain each comprising LCDRs 1 to 3 of the VL chain of the MRK16 antibody, and the second VH chain comprises LCDRs 1 to 3 of the 5F9 antibody or B6H12 28. The bispecific antibody molecule of claim 27, comprising HCDRs 1 to 3 of the VH chain of the antibody.

41. the first VH chain comprises the VH chain of the B1.225 antibody listed in Table 2, and the identical VL chain comprises the VL chain of the B1.225 antibody listed in Table 2, 41. The dual antibody of claim 40, wherein the dual antibody comprises a VL chain of the K16 antibody, and the second VH chain comprises a VH chain of the 5F9 antibody. Specificity antibody molecule.

42. The antibody molecule or bispecific antibody according to any of the preceding claims, wherein the antibody comprises a humanized light chain. sexual antibody molecule.

43. The antibody molecule or bispecific antibody according to any of the preceding claims, wherein the antibody comprises a humanized heavy chain. sexual antibody molecule.

44. 10. A method for treating cancer in a subject, comprising administering to a subject a compound according to any one of the preceding claims. and a method for administering the antibody or bispecific antibody molecule to the subject. An antibody molecule or a bispecific antibody molecule, comprising administering to said antibody molecule or bispecific antibody molecule.

45. The method 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 45. The antibody molecule of claim 44, comprising an inhibitor, an immunotherapeutic agent, or a combination thereof. Bispecific antibody molecule.

46. The at least one additional active agent is a chemotherapeutic agent, and optionally, the chemotherapeutic agent is a 46. ​​The compound of claim 45, which is a benzodiazepine, a vinca alkaloid, or an anthracycline. An antibody molecule or a bispecific antibody molecule.

47. the subject to be treated has a cancer that has been determined to be resistant to treatment with said chemotherapeutic agent; 47. An antibody molecule or bispecific antibody molecule according to claim 46.

48. An antibody molecule according to any one of claims 1 to 21 or any one of claims 22 to 43 A method for treating cancer, comprising administering to a subject a therapeutically effective amount of the bispecific antibody molecule described in claim 1. How to treat an elephant.

49. The method comprises administering to said patient an antibody molecule or bispecific antibody molecule in combination with at least one additional active agent. and administering an isomeric antibody molecule to the subject, wherein the at least one additional active agent is a chemotherapeutic agent. , an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. Item 49. The method according to item 48.

50. The at least one additional active agent is a chemotherapeutic agent, and optionally, the chemotherapeutic agent is a 50. The compound of claim 49, wherein the compound is a benzodiazepine, a vinca alkaloid, or an anthracycline. method.

51. the subject being treated has a cancer classified as resistant to said chemotherapeutic agent. Item 50. The method according to item 50.

52. An antibody molecule according to any one of claims 1 to 21 or any one of claims 22 to 43 one or more nucleic acids comprising one or more sequences encoding the bispecific antibody molecule of claim 1. acid.

53. 53. One or more recombinant expression vectors comprising one or more nucleic acids of claim 52. -.

54. 54. A host cell genetically modified with one or more recombinant expression vectors of claim 53. Cell.

55. Chimeras containing MDR1-binding domains, transmembrane domains, and intracellular signaling domains an immune effector cell comprising an MDR1-binding domain comprising an antigen receptor (CAR), the MDR1-binding domain comprising: Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 and / or light chain CDRs (LCDRs).

56. A method for inhibiting the efflux activity of MDR1 expressed by a living cell, comprising: The antibody molecule according to any one of claims 1 to 21 or the antibody molecule according to any one of claims 22 to 43 The method comprises contacting a subject with the bispecific antibody molecule of claim 1.

57. 57. The method of claim 56, further comprising contacting the cells with an inhibitor of MDR1-mediated efflux. Law.

58. 58. The method of claim 56 or 57, further comprising contacting the cells with a chemotherapeutic agent. method.

59. 59. The method of any one of claims 56 to 58, wherein the cells are cancer cells.

60. 60. The method of claim 59, wherein the cancer cells are multidrug resistant cancer cells.

61. A method for assaying the expression of MDR1 on the cell surface of a cell, comprising:

48. A method comprising contacting a subject with the antibody molecule of any one of claims 47.

62. 62. The method of claim 61, wherein the antibody is detectably labeled.

63. 1. A method for diagnosing a disease or condition associated with MDR1 expression in a subject, comprising: A tissue or cell sample from a subject is subjected to the immunoprecipitation of an antibody molecule according to any one of claims 1 to 47. The method of claim 1, wherein the .alpha.-.alpha.

64. The antibody molecule has the VH and VL chain configuration shown for the antibody molecule in FIG.

64. The method of claim 63, comprising:

65. An antibody that specifically binds to multidrug resistance protein 1 (MDR1) for use as a pharmaceutical. The antibody variable heavy chain (VH) and variable light chain (VL) regions of the antibody shown in Table 2 are overlapping. an antibody comprising light chain complementarity determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3); An antibody that competes for binding to R1.

66. Specific binding to multidrug resistance protein 1 (MDR1) for the manufacture of a medicament for the treatment of cancer Use of an antibody, said antibody comprising a variable heavy chain (VH) region and a variable heavy chain (VH) region of an antibody listed in Table 2. The pair of variable light chain (VL) regions contains heavy chain complementarity determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1 to 3) for binding to MDR1.

67. Multidrug resistance protein 1 (MDR1) for use in a method of treating cancer in a subject 1. A pharmaceutical composition comprising an antibody that specifically binds to a nucleotide sequence of a nucleotide sequence selected from the group consisting of nucleotides 1 and 2, wherein the antibody is an antibody listed in Table 2. The heavy chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) of the pair of variable heavy chain (VH) and variable light chain (VL) regions of competes for binding to MDR1 with an antibody comprising light chain CDRs 1-3 (LCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3), The method comprises administering the antibody to the subject.

68. Antibody molecules that specifically bind to multidrug resistance protein 1 (MDR1) for pharmaceutical use The antigen-binding site of the antibody molecule is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 sex-determining regions 1–3 (HCDRs 1–3) and light chain CDRs 1–3 (LCDRs 1–3); HCDRs 1-3 of the VH region of an antibody listed in Table 2; or LCDRs 1 to 3 of the VL regions of the antibodies listed in Table 2 Including, antibody molecule.

69. Specific binding to multidrug resistance protein 1 (MDR1) for the manufacture of a medicament for the treatment of cancer Use of an antibody molecule, wherein the antigen-binding site of said antibody molecule comprises: Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 Sex-determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3); antibodies listed in Table 2 or LCDRs 1-3 of the VL region of an antibody listed in Table 2, use.

70. Multidrug resistance protein 1 (MDR1) for use in a method of treating cancer in a subject a pharmaceutical composition comprising an antibody that specifically binds to a target polypeptide, wherein the antigen-binding site of the antibody molecule is Heavy chain complementarity of pairs of variable heavy (VH) and variable light (VL) chains of antibodies listed in Table 2 Sex-determining regions 1-3 (HCDRs 1-3) and light chain CDRs 1-3 (LCDRs 1-3); antibodies listed in Table 2 or LCDRs 1 to 3 of the VL region of an antibody described in Table 2, The method includes administering the antibody to the subject. Pharmaceutical compositions.

71. 22. An antibody molecule according to claim 1, 7 or 21 for use as a medicament.

72. 22. Use of an antibody molecule according to claim 1, 7 or 21 for the manufacture of a medicament for the treatment of cancer.

73. 22. The method of claim 1, 7 or 21 for use in a method of treating cancer in a subject. wherein the method comprises administering the antibody to the subject. A pharmaceutical composition comprising:

74. Multidrug resistance protein 1 (MDR1) and tumor-associated antigens (TAA) for pharmaceutical use a bispecific antibody molecule that binds to a first variable heavy (VH) chain and a second variable light (VL) chain; ) chain, and a second VH chain, each of said VL chains comprising an antigen-binding site for MDR1; The first VH chain comprises an antigen-binding site for MDR1, and the second VH chain comprises an antigen-binding site for TAA. and wherein the second VH chain binds to the TAA when paired with one of the light chains. A bispecific antibody molecule.

75. Multidrug resistance protein 1 (MDR1) and tumor-associated antigens for the manufacture of a medicament for the treatment of cancer The use of a bispecific antibody molecule that binds to a TAA, said antibody molecule binding to two identical potential a variable light (VL) chain, a first variable heavy (VH) chain, and a second VH chain, each of the VL chains being an MDR1 the first VH chain comprises an antigen-binding site for MDR1, The second VH chain comprises an antigen-binding site for the TAA, and the second VH chain binds to one of the light chains. The use of a nucleotide sequence that binds to the TAA when paired with one of the nucleotide sequences.

76. Multidrug resistance protein 1 (MDR1) for use in a method of treating cancer in a subject and a pharmaceutical composition comprising a bispecific antibody molecule that binds to a tumor-associated antigen (TAA), The antibody molecule comprises two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain. wherein each of the VL chains comprises an antigen-binding site for MDR1, and the first VH chain comprises an antigen-binding site for MDR1. the second VH chain comprises an antigen-binding site for said TAA, a second VH chain that binds to the TAA when paired with one of the light chains, and the method further comprises: A pharmaceutical composition comprising administering the antibody to the subject.

77. 75. The bispecific molecule of claim 74, wherein the bispecific molecule comprises LCDRs 1 to 3 of the VL region of an antibody listed in Table 2.

77. The bispecific molecule of claim 75, the use of claim 75, or the pharmaceutical composition of claim 76.

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