Excretory pump - cancer antigen multi-specific antibody and compositions, reagents, kits and methods related thereto

Abstract

Multispecific antibodies that target both cellular efflux pumps and cancer-associated antigens are provided, as well as pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits that contain or encode such multispecific antibodies. Methods of treating a subject for cancer are also provided, comprising administering to the subject a multispecific antibody that targets both cellular efflux pumps and cancer-associated antigens. Methods of producing the described multispecific antibodies and related reagents, including recombinant cell lines useful in the methods, are also provided, as well as methods of producing such recombinant cell lines.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 967,478, filed on January 29, 2020, and U.S. Provisional Patent Application No. 62 / 828,044, filed on April 2, 2019, the entire contents of which are incorporated herein by reference.

Background Art

[0002] Drug resistance is a well - known phenomenon that occurs when a disease becomes resistant to drug treatment and is a major and increasing problem in various fields of medicine, including oncology. Some methods of drug resistance are disease - specific, but there are also evolutionarily conserved methods such as drug efflux observed in microorganisms and human drug - resistant cancers. Many types of cancer are initially sensitive to chemotherapy but can develop resistance over time through these mechanisms as well as other mechanisms such as DNA mutations and metabolic changes that promote inhibition, degradation, and enhanced efflux of drugs.

[0003] Efflux pumps are proteins expressed by virtually all living cells and have evolved to naturally expel various compounds from the cell. Members of the ATP-binding cassette (ABC) transporter family of proteins are examples of EPs that enable drug efflux and are important and well-studied regulators in the cell membranes of healthy cells. Although the structures of transporters vary from protein to protein (for example, there are 49 known members in the human ABC family), they are all classified by the presence of two different domains: a highly conserved nucleotide-binding domain and a more variable transmembrane domain. Multidrug resistance protein 1, encoded by the ATP-binding cassette subfamily B member 1 (ABCB1) gene, was the first of these to be identified and has been extensively studied. Normal expression of MDR1 increases in certain tissues (such as the colon, liver, kidney, etc.) when they become neoplastic, and the increase in expression in response to treatment with certain chemotherapeutic agents indicates that overexpression of MDR1 by both endogenous and exogenous mechanisms is at work.

[0004] EPs enable cells and tumors to develop resistance to chemotherapeutic agents. Such resistance is often associated with increased efflux of therapeutic molecules from resistant cells. This chemotherapeutic resistance is called multi-drug resistance (MDR) when multiple chemotherapeutic agents are involved. Various small molecule inhibitors that target and inhibit EPs have been developed, but they have not been successful in human clinical settings for various reasons. Among those reasons is that they tend to penetrate and affect all cells in the body (including healthy cells that use EPs for the efflux of naturally occurring cytotoxins) regardless of the function of the cells or their efflux pumps.

[0005] Among cancer patients in whom the majority of a metastatic cancer cell population has been killed and removed from the patient by use of chemotherapy, it is not uncommon for a drug-resistant cancer cell population to arise and expand without responding to new treatment by previous therapies. In most cases, another drug having a different mechanism of action is applied, but again, additional drug-resistant cell populations and / or tumors will arise.

SUMMARY OF THE INVENTION

[0006] An anti-MDR1 antibody that can be used as a multi-specific antibody targeting both MDR1 and a tumor-associated antigen (TAA), and pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such multi-specific antibodies are provided. This multi-specific antibody includes a common variable light (VL) chain containing an antigen-binding site for MDR1, a first variable heavy (VH) chain containing an antigen-binding site for MDR1, and a second VH chain containing an antigen-binding site for TAA. Also provided is a method of treating a subject for cancer, including administering to the subject a multi-specific antibody targeting both MDR1 and TAA. The treatment can include administering the multi-specific antibody alone or administering the multi-specific antibody and a chemotherapeutic agent. Also provided are methods of making the described multi-specific antibodies and related reagents, including recombinant cell lines useful in the methods of the invention and methods of making them.

[0007] The bispecific antibodies provided herein bind to cancer cells expressing both MDR1 and TAA, but show reduced binding to non-cancer cells expressing MDR1 and / or TAA. In other words, the bispecific antibodies provided herein bind with low affinity to (1) TAA-expressing cells with low or no MDR1 expression, and (2) MDR1-expressing cells with low or no TAA expression, and bind with high affinity to cancer cells expressing at least one or both of MDR1 and CD47 at relatively high levels, i.e., at levels higher than normal cells.

[0008] Also disclosed herein are anti-MDR1 antibodies having a reduced affinity for MDR1 compared to the anti-MDR1 antibody 15D3, and anti-CD47 antibodies having a reduced affinity for CD47 compared to the anti-CD47 antibody 5F9. These antibodies, when co-administered, find use in the treatment of cancer in a subject and increase the chemosensitivity of cancer cells to chemotherapy.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0010] [Definitions] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that maintain specific binding to an antigen (including but not limited to Fab, Fv, scFv, and Fd fragments), chimeric antibodies, humanized antibodies, single-chain antibodies (including antibodies containing only the heavy chain, such as VHH camel antibodies), bispecific antibodies, and fusion proteins containing the antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be detectably labeled, for example, by radioisotopes, enzymes that produce detectable products, fluorescent proteins, etc. Antibodies can be further conjugated to other moieties, such as members of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be bound to a solid support, including but not limited to polystyrene plates or beads, etc. The term also encompasses Fab', Fv, F(ab')2, and other antibody fragments that maintain specific binding to an antigen, as well as monoclonal antibodies. Antibodies can be monovalent or divalent. The antibodies used herein can be used, for example, to assay the expression of target antigen(s) on the cell surface in a cell sample or tissue sample derived from a patient.

[0011] An "antibody fragment" includes a portion of an intact antibody and, for example, includes the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)), single-chain antibody molecules (including antibodies containing only the heavy chain, such as VHH camel antibodies), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment (a designation that reflects the ability to readily crystallize). Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and can still cross-link antigens.

[0012] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen-binding site. This region consists of a dimer in which one heavy-chain variable domain and one light-chain variable domain are tightly non-covalently bound. In this arrangement, the three CDRs of each variable domain interact to form a V H -V L -dimer surface that defines the antigen-binding site. Collectively, these six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three CDRs specific for the antigen) has the ability to recognize and bind the antigen and can form an antigen-binding site, but with lower affinity than the complete binding site containing the three CDRs of each variable domain.

[0013] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by the addition of several residues at the carboxyl terminus of the heavy-chain CH1 domain that includes one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with an intervening hinge cysteine. Other chemical couplings of antibody fragments are also known.

[0014] The "light chains" of antibodies (immunoglobulins) from vertebrate species can be assigned to one of two distinct types called kappa and lambda based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0015] "Single-chain Fv", "sFv", or scFv antibody fragments contain the V H and V L domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the sFv to form the desired structure for antigen binding, VH and VL domains. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0016] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment containing a heavy-chain variable domain (V H -V L ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains on another chain to create two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0017] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances, and is expressed as the dissociation constant (Kd). The affinity can be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold or more greater than the affinity of the antibody for an irrelevant amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactivity" and "preferentially binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0018] The term "binding" means the direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic bonds and / or hydrogen-bonding interactions, and includes interactions such as salt bridges and hydrogen bridges. The MDR1-specific antibody specifically binds to an epitope within the MDR1 polypeptide. Nonspecific binding means binding at an affinity of less than about 10 -7 M (for example, an affinity at 10 -6 M, 10 -5 M, 10 -4 M, etc.).

[0019] As used herein, the term "CDR" or "complementary determining region" is intended to mean non - contiguous antigen - binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest”(1991); by Chothia et al., J. Mol. Biol. 196:901 - 917 (1987); and MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996), and the definitions therein include amino acid residues that overlap or are subsets when compared to each other. Nevertheless, the application of any of the definitions for representing CDRs of an antibody or grafted antibody or their variants is intended to be within the scope of the terms defined and used herein. The amino acid residues encompassing the CDRs defined by each of the above - cited references are shown in Table 1 below for comparison.

[0020] [Table 1]

[0021] As used herein with respect to antibody variable regions, the term "framework" is intended to mean all amino acid residues outside of the CDR regions within the variable region of an antibody. The variable region framework is generally a discontinuous amino acid sequence of about 100 to 120 amino acids in length, but is intended to refer only to the amino acids outside of the CDRs. The term "framework region" as used herein is intended to mean each domain of the framework separated by the CDRs. The VH chain can include three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, the VL chain can include three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0022] As used herein, the term "antibody" encompasses 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 designated CH1, CH2, and CH3. The light chain constant region is composed of one domain designated CL. The variable regions of the heavy and light chains include the binding regions that interact with an antigen. The constant region of an antibody typically mediates the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system. The term "antibody" includes immunoglobulins of IgA, IgG, IgE, IgD, IgM, and their subtypes. In certain embodiments, the antibody of interest is of the IgG isotype, for example, IgG1.

[0023] As used herein, the term "immunoglobulin" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes; and numerous immunoglobulin variable region genes. A full-length immunoglobulin light chain (about 25 kD or 214 amino acids) is encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region at the C-terminus. A full-length immunoglobulin heavy chain (about 50 kD or 446 amino acids) is encoded by a variable region gene at the N-terminus (about 116 amino acids) and one of the other constant region genes at the C-terminus, for example gamma (encoding about 330 amino acids). In certain embodiments, the subject antibody comprises a complete immunoglobulin comprising a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0024] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to an antigen. Examples of binding fragments include the following: (i) Fab fragment (a monovalent fragment consisting of, for example, VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region); (iii) Fd fragment (one containing VH and CH1 domains, for example, consisting of them); (iv) Fv fragment (one containing VH and VL domains of a single arm of an antibody, for example, consisting of them); (v) dAb fragment (one containing VH domain, for example, consisting of VH domain); (vi) isolated CDR; (vii) single-chain Fv (scFv) (including those in which VH and VL domains of a single arm of an antibody are linked by a synthetic linker using recombinant means so that VH and VL domains pair to form a monovalent molecule, for example, consisting of it); (viii) diabody (one containing two scFvs in which VH domains and VL domains are linked so as not to pair to form a monovalent molecule, for example, consisting of two such scFvs; each VH of the scFv pair forms a pair with the VL domain of the other scFv to form a bivalent molecule).

[0025] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portion of the heavy and / or light chain is derived from a different source or species.

[0026] A "human antibody" is an antibody produced by a human or human cell, or one having an amino acid sequence corresponding to an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0027] The "human consensus framework" is a framework (FR) that represents the amino acid residues most commonly occurring in the selection of human immunoglobulin variable light chain (VL) or variable heavy chain (VH) framework sequences. Generally, the selection of the VL or VH sequence of a human immunoglobulin occurs from a subgroup of variable domain sequences. Generally, this subgroup of sequences is a subgroup such as those in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for VL, this subgroup is subgroup kappa I in the above Kabat et al. In one embodiment, for VH, this subgroup is subgroup III in the above Kabat et al.

[0028] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human CDRs and amino acid residues derived from human frameworks (FRs). At least a portion of the constant region of the humanized antibody is derived from a human antibody. In a preferred embodiment of the antibody molecules disclosed herein, the constant region is from a human IgG antibody such as human IgG1. In a preferred embodiment, the antibody molecules disclosed herein comprise a heavy chain comprising the variable heavy chain region provided herein and a human IgG1 constant region having the amino acid sequence provided in UniProt: P01857-1 version 1. In a preferred embodiment, the antibody molecules disclosed herein comprise a light chain comprising the variable light chain region provided herein and a human light chain constant region. In a preferred embodiment, the human light chain constant region is a human kappa light chain constant region. In certain embodiments, the human IgG1 heavy chain constant region present in the subject antibody may contain mutations, such as substitutions for modulating Fc function. For example, the LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation can be introduced to reduce antibody-dependent cell cytotoxicity (ADCC). The numbering of the substitutions is based on the EU numbering system. The "EU numbering system" or "EU index" is generally used when referring to residues within the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0029] The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0030] The term "epitope" refers to the region of an antigen that is recognized by the immune system (e.g., an antibody, B cell, or T cell). For example, an epitope is a specific region of an antigen to which an antibody binds.

[0031] An "isolated" antibody is one that has been identified and separated and / or recovered from a component in its natural environment. The contaminant components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99% by weight of the antibody as measured by the Lowry method, (2) to a sufficient extent to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody includes an antibody in situ within a recombinant cell since at least one component of the antibody's natural environment is absent. In some instances, an isolated antibody is prepared by at least one purification step.

[0032] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or cell destruction. A "chemotherapeutic agent" may also be referred to as an "antineoplastic agent" and is a cytotoxic agent used to treat cancer or other diseases or disorders.

[0033] As used herein, terms such as "treatment" and "treating" mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in the sense of completely or partially preventing a disease or its symptoms, and / or can be therapeutic in the sense of partially or completely curing a disease and / or its adverse effects resulting therefrom. "Treatment" as used herein encompasses any treatment of a disease in a mammal, including a human, and includes the following: (a) preventing a disease from occurring in a subject who may have a predisposition to the disease but has not yet been diagnosed as having the disease; (b) suppressing the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing regression of the disease.

[0034] The terms "individual", "subject", "host" and "patient", which are used interchangeably herein, refer to mammals including, but not limited to, rodents (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats).

[0035] "Therapeutically effective amount" or "effective amount" refers to the amount of a target-specific antibody sufficient to effect such treatment for a disease when administered to a mammal or other subject for treating the disease. The "therapeutically effective amount" varies depending on the antibody, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0036] The term "refractory" as used herein refers to a disease or condition that does not respond to treatment. With respect to cancer, "treatment-refractory cancer" as used herein refers to cancer that does not respond to treatment. Treatment-refractory cancer may show resistance at the start of treatment or may become resistant during treatment. Treatment-refractory cancer is also referred to as resistant cancer.

[0037] "Biological sample" encompasses various sample types obtained from an individual and can be used in diagnostic or monitoring assays. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy samples or tissue cultures, or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in some way after their procurement, such as by treatment with reagents, solubilization, or concentration of specific components such as polynucleotides. The term "biological sample" includes clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue samples.

[0038] The percent identity between a pair of sequences can be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region (including gaps). In identity scoring, only exact matches are counted and the degree of similarity between amino acids is not considered. The length includes only internal gaps and does not include gaps at the ends of the sequences. Percent identity = (matches x 100) / length of aligned region (including gaps)

[0039] The phrase "conservative amino acid substitution" refers to substitutions of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; 6) D, E. Conservative amino acid substitutions can preserve the activity of a protein by replacing an amino acid(s) in the protein with an amino acid having a side chain with similar acidic, basic, charge, polarity, or size.

[0040] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to convey protein-coding information to a host cell.

[0041] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains a nucleic acid sequence that (along with the host cell) induces and / or controls the expression of one or more heterologous coding regions operably linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, where introns are present, RNA splicing of the operably linked coding region.

[0042] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex or signal transduction via the signal transduction domain of an appropriate NK receptor or CAR. Stimulation can mediate a change in the expression of particular molecules.

[0043] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides a cytoplasmic signaling sequence that regulates the activation of the immune cell in a stimulatory manner with respect to at least one aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated, for example, by the binding of the TCR / CD3 complex to an MHC molecule loaded with a peptide, which results in the mediation of a T cell response (including but not limited to proliferation, activation, differentiation, etc.). The primary cytoplasmic signaling sequence that acts in a stimulatory manner (also referred to as the "primary signaling domain") may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing cytoplasmic signaling sequences particularly useful in the present invention include those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12, but are not limited thereto.

[0044] The term "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand and thereby mediates a co-stimulatory response (such as, but not limited to, proliferation) by the T cell. Co-stimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that contribute to an efficient immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0045] The term "autologous" refers to a substance derived from the same individual into which the substance is later reintroduced.

[0046] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates signals that promote the immune effector function of a CAR-containing cell (e.g., a CAR-T cell). For example, examples of immune effector functions in CAR-T cells include cytolytic activity and helper activity, which include the secretion of cytokines.

[0047] As used herein, the term "immune effector cell" refers to a cell involved in an immune response, for example, in promoting an immune effector response. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0048] Guidance for substitutions, insertions or deletions may be derived from an alignment of the amino acid sequences of proteins from different species or from a consensus sequence based on multiple proteins having the same or similar functions.

[0049] [Detailed Description] An anti-MDR1 antibody that can be used as a bispecific antibody targeting both MDR1 and a tumor-associated antigen (TAA), as well as a pharmaceutical composition, nucleic acid, recombinant expression vector, cell, and kit comprising or encoding such a bispecific antibody are provided. The bispecific antibody comprises a common variable light (VL) chain comprising an antigen-binding site for MDR1, a first variable heavy (VH) chain comprising an antigen-binding site for MDR1, and a second VH chain comprising an antigen-binding site for TAA. Also provided is a method of treating a subject for cancer, comprising administering to the subject a bispecific antibody targeting both MDR1 and TAA. Treating can comprise administering the bispecific antibody alone or administering the bispecific antibody and a chemotherapeutic agent. Also provided are methods of making the described bispecific antibodies and related reagents, which include recombinant cell lines useful in the methods of the invention and methods of making such recombinant cell lines.

[0050] The bispecific antibodies provided herein bind to cancer cells expressing both MDR1 and TAA, but show reduced binding to non-cancer cells expressing MDR1 and / or TAA. In other words, the bispecific antibodies provided herein bind with low affinity to (1) cells that express TAA but have low or no MDR1 expression, and (2) cells that express MDR1 but have low or no TAA expression, and bind with high affinity to cancer cells expressing at least one or both of MDR1 and CD47 at relatively high levels (i.e., levels higher than normal cells).

[0051] Also disclosed herein are anti-MDR1 antibodies with reduced affinity for MDR1 compared to the anti-MDR1 antibody 15D3, and anti-CD47 antibodies with reduced affinity for CD47 compared to the anti-CD47 antibody 5F9. These antibodies find use in the treatment of cancer in a subject when co-administered with chemotherapy to increase the chemosensitivity of cancer cells to the chemotherapy.

[0052] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, and such embodiments can of course vary. Also, the scope of the present invention is limited only by the appended claims, and it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0053] When numerical ranges are provided, each intervening value, to the extent not otherwise clearly indicated by the context, is included within the invention to within one tenth of the unit of the lower limit, between the upper and lower limits of that range and other stated or intervening values within that stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the invention, subject to any specifically excluded limitations within the stated range. When the stated range includes one or both of the upper and lower limit values, ranges excluding either or both of those values are also included in the invention.

[0054] A particular range is presented by a numerical value following the term "about". The term "about" is used herein to provide a textual basis for the exact number following the term, as well as for a number that is close to or an approximation of the number following the term. In determining whether a number is close to or an approximation of a specifically recited number, a number that is close to or an approximation but not specifically recited may be considered to be a number that is substantially equivalent to the specifically recited number in the context in which it is presented.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but representative exemplary methods and materials are described herein.

[0056] All publications and patents cited in this specification are hereby incorporated by reference into this specification as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, to disclose and describe the methods and / or materials relevant to the places where such publications are cited. The citation of a publication is for disclosure prior to the filing date of this application and should not be construed as an admission that the present invention has the right to precedence over such a publication. Further, the provided publication date may be different from the actual publication date that needs to be independently verified.

[0057] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Further, it should be noted that the claims can be drafted to exclude optional elements. Accordingly, this statement is intended to serve as a textual basis for the use of exclusive terms such as "solely", "only", etc. in relation to the recitation of claim elements, or for the use of "except for" limitations.

[0058] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with any of the features of some other embodiments without departing from the scope or spirit of the present invention. The described methods can be performed in the order of the described events or in any other order that is logically possible.

[0059] The methods and compositions are described, or will be described, with functional descriptions for grammatical flow, but the claims should not be construed as necessarily limited by a "means" or "step" limitation configuration, unless expressly formulated based on 35 U.S.C. § 112(f), and should be given the meaning of the definitions provided by the claims under the doctrine of equivalents and the full scope of equivalents, and it should be clearly understood that when the claims are expressly formulated based on 35 U.S.C. § 112(f), the full statutory equivalents based on 35 U.S.C. § 112(f) should be given.

[0060] antibody [bispecific antibody] The present disclosure provides a bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and a tumor-associated antigen (TAA), the antibody molecule comprising two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain, wherein each VL chain comprises an antigen-binding site for MDR1, the first VH chain comprises an antigen-binding site for MDR1, the second VH chain comprises an antigen-binding site for TAA, and the second VH chain binds to TAA when paired with one of the light chains. The bispecific antibody molecule binds to cancer cells that express both MDR1 and TAA, but exhibits reduced binding to non-cancer cells that express MDR1 and / or TAA. In other words, the bispecific antibody provided herein binds with low affinity to (1) TAA-expressing cells with low or absent MDR1 expression, and (2) MDR1-expressing cells with low or absent TAA expression, and binds with high affinity to cancer cells that express at least one or both of MDR1 and CD47 at relatively high levels, i.e., levels higher than normal cells.

[0061] By a relatively high level is meant an expression that is at least 1.5-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more) the expression level in normal cells of the same type as the cancer cells. By a reduced affinity is meant a binding affinity that is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, or more) compared to the binding of the antibody molecule to normal cells of the same type as the cancer cells. Reduced affinity includes the absence of detectable binding.

[0062] The term “antibody molecule” includes antibodies as defined herein and antigen-binding fragments thereof. In certain embodiments, the antibody molecule includes two variable light (VL) chains and two variable heavy (VH) chains. In certain embodiments, the antibody molecule also includes the constant regions of the heavy and light chains. The heavy chain constant region and the light chain constant region can be derived from human antibodies, such as human IgG1 antibodies. The human IgG1 heavy chain (HC) constant region can be modified to include mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC). In addition or alternatively, the two VH chains can each be linked to a different human IgG1 HC constant region such that the individual human IgG1 HC constant regions have substitutions that are favorable for dimer formation between different human IgG1 HC constant regions. Such HC regions are described in more detail herein. In certain embodiments where the antibody molecule is a bispecific antibody molecule, one of the human IgG1 HC constant regions includes substitutions that introduce one or more amino acids having a positively charged side chain and the other human IgG1 HC constant region includes substitutions that introduce one or more amino acids having a negatively charged side chain, which can promote dimer formation between the two different HCs.

[0063] In certain embodiments, the antigen-binding sites of the two VL chains include light chain CDRs (LCDRs) of a VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK Here X 1 is N, Q, or S.

[0064] In certain embodiments, the two VL chains comprise LCDR 1-3 of a VL chain having the following sequences: (i) DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK; (ii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; or (iii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK.

[0065] In some embodiments, the antibodies described herein comprise: (i) two VL chains comprising the LCDRs of a VL chain having the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK and a first VH chain comprising the HCDRs of a VH chain having the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS does not include.

[0066] In certain embodiments, the CDRs of the VL and VH light chains can be defined based on the Kabat nomenclature.

[0067] In certain embodiments, i) LCDR1 comprises the sequence RSSQSIVHSTGX 1 TYLE, (ii) LCDR2 comprises the sequence KISNRFS, and (iii) LCDR3 comprises the sequence FQASHFPRT, where X 1 is N, Q, or S. These LCDRs are based on the Kabat nomenclature.

[0068] In certain embodiments, the two VL chains are humanized. In certain embodiments, the two VL chains are humanized to include framework regions from human antibodies.

[0069] In certain embodiments, the two VL chains comprise sequences that are at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the following sequences: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK; DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; or DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK.

[0070] In certain embodiments, the bispecific antibody molecule each has the following sequences: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK comprises two VL chains comprising

[0071] In certain embodiments, the bispecific antibody comprises a light chain comprising the VL chain described herein and a light chain constant region. The light chain constant region can be a human immunoglobulin kappa chain constant region having the amino acid sequence described in UniProtKB / Swiss-Prot: P01834.2.

[0072] In certain embodiments, the bispecific antibody molecule comprises a first VH chain, which VH chain has the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS and comprises heavy chain CDRs 1-3 (HCDR 1-3) of a VH chain having the sequence, where X 2 is N, Q or S.

[0073] In certain embodiments, the first VH chain comprises HCDR 1-3 of a VH chain having the following sequence. (i) EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or (ii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or (iii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0074] In certain embodiments, the HCDR 1-3 of the VH chain are defined based on the Kabat nomenclature.

[0075] In certain embodiments, the first VH chain comprises: (i) HCDR1 comprising the sequence RYTMS; (ii) HCDR2 comprising the sequence TISSGGGX 2 TYYPDSVKG; and (iii) HCDR3 comprising the sequence YGAGDAWFAY, where X 2 is N, Q or S. These HCDRs are based on the Kabat nomenclature.

[0076] In certain embodiments, the first and / or second VH chains are humanized. In certain embodiments, the VH chain is humanized to include a framework region from a human antibody.

[0077] In certain embodiments, the first VH chain comprises the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0078] In certain embodiments, the first VH chain comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0079] In certain embodiments, the bispecific antibody comprises a first heavy chain comprising the first VH chain as described herein, and a human IgG1 heavy chain constant region.

[0080] In certain embodiments, the second VH chain of the bispecific antibody is derived from a monospecific antibody molecule that binds to a TAA, where the affinity of the bispecific antibody molecule for the TAA is at least 2-fold lower (e.g., at least 3-fold lower, at least 4-fold lower, at least 5-fold lower) than the affinity of the monospecific antibody molecule from which its VH chain is derived for the TAA. The affinities of the bispecific antibody and the monospecific antibody are measured using the same assay. Any suitable method for measuring antibody affinity can be utilized. In certain embodiments, the affinity can be measured by calculating the equilibrium constant for the reversible binding of the antibody to the antigen, expressed as the dissociation constant (Kd). In certain embodiments, the Kd can be measured by ELISA.

[0081] In certain embodiments, the second VH chain of the bispecific antibody is derived from a monospecific antibody molecule that binds to a TAA, where the half-maximal effective concentration (EC50) of the bispecific antibody molecule for the TAA is at least 2-fold higher (e.g., at least 3-fold higher, at least 4-fold higher, at least 5-fold higher) than the EC50 of the monospecific antibody molecule from which its VH chain is derived for the TAA. The EC50s of the bispecific antibody and the monospecific antibody are measured using the same assay. Any suitable method for measuring the EC50 of an antibody can be utilized. The concentration that shows half of the maximum response (e.g., half of the maximum fluorescence intensity) is measured as the EC50.

[0082] The EC50 of the test antibody can be measured by flow cytometry or ELISA. For example, flow cytometry involves contacting cells expressing an antigen (e.g., human wild-type MDR1 or mutant MDR1) with the antibody in flow cytometry buffer (where the antibody is serially diluted), and incubating at room temperature or 4°C for a time sufficient for the antibody to bind to the cells (e.g., 10 minutes to 1 hour). After incubation, the cells may optionally be washed to remove non-specifically bound antibody, and / or the cells may be contacted with a fluorescently labeled secondary antibody that specifically binds to the test antibody. After incubation, the fluorescently labeled secondary antibody may be removed and the cells washed. The washed cells can be sorted by flow cytometry, and the number of cells bound to the fluorescently labeled secondary antibody can be counted. The concentration that provides half of the maximum response (e.g., half of the maximum fluorescence intensity) is measured as the EC50. In a variation of the flow cytometry assay, the cells can be 293T cells that overexpress MDR1.

[0083] The TAA can be any antigen known to be overexpressed in cancer cells. For example, the TAA can be an antigen that is not expressed at detectable levels in normal cells but is expressed in cancer cells, where the normal and cancer cells are of the same cell type, e.g., epithelial cells. For example, the TAA can be a neoantigen, which is a class of tumor antigens that result from tumor-specific mutation(s) that alter the amino acid sequence of the encoded protein compared to the amino acid sequence of the unmutated protein. In other embodiments, the TAA is an antigen that is expressed in normal cells but is expressed at higher levels in cancer cells. In certain embodiments, the TAA can be CD47.

[0084] [Anti-MDR1 and anti-CD47 bispecific antibody] In certain embodiments, the bispecific antibody molecule binds to CD47 and the second VH chain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS comprises the HCDR of the VH chain containing

[0085] In certain embodiments, the second VH chain comprises HCDR1 comprising the sequence NYNMH, HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD, and HCDR3 comprising the sequence GGYRAMDY. HCDR 1-3 are defined according to Kabat nomenclature.

[0086] In certain embodiments, the second VH chain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS and comprises a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, or at least 95% identical) to the following:

[0087] In certain embodiments, the second VH chain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS and comprises

[0088] In certain embodiments, the bispecific antibody that binds to MDR1 and CD47 may comprise a humanized VH chain comprising an antigen-binding site for CD47 having the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS; EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS; or QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS。

[0089] Further aspects of the bispecific antibodies are described elsewhere in the present application and include variations of the sequences disclosed herein, humanized versions, and / or substitutions in the Fc region that promote the formation of heterodimers between the first and second VH chains.

[0090] [Anti-MDR1 and anti-PD-L1 bispecific antibody] In certain aspects, the TAA can be programmed cell death ligand 1 (PD-L1). PD-L1 is also known as cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7-H1). In one aspect, the bispecific antibody molecule that binds to MDR1 and PD-L1 comprises a common light chain and a first VH chain, as described in the previous section, and the second VH chain has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS comprises HCDR 1-3 of the VH chain.

[0091] The HCDR 1-3 defined according to the Kabat nomenclature are as follows. HCDR1: DSWIH HCDR2: WISPYGGSTYYADSVKG HCDR3: RHWPGGFDY

[0092] The second VH chain of the bispecific antibody that binds to MDR1 and PD-L1 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS may have an amino acid sequence with at least 80%, at least 90%, at least 95%, or 100% identity thereto.

[0093] The second VH chain of the bispecific antibody that binds to MDR1 and PD-L1 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK It may be present in the heavy chain having an amino acid sequence with at least 80%, at least 90%, at least 95%, or 100% identity to

[0094] As described elsewhere herein, at least one, two, or all of the VL chain, the first VH chain, and the second VH can be humanized. Further, the Fc region of the VH chain may contain substitutions that increase heterodimerization between the first and second VH chains. In certain embodiments, the first heavy chain can be humanized and contain the charge pair substitutions K392D and K409D, and the second heavy chain can contain the charge pair substitutions E356K and D399K.

[0095] [Anti-MDR1 and anti-EGFR bispecific antibody] In certain embodiments, the TAA can be the epidermal growth factor receptor (EGFR). The HCDR 1-3 of the second VH chain containing the antigen-binding site for EGFR can be derived from the VH chain of the anti-EGFR antibody necitumumab or cetuximab. The heavy chain of necitumumab has the following sequence. QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0096] The heavy chain of cetuximab has the following sequence. QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0097] In a first aspect, the anti-MDR1 anti-EGFR bispecific antibody comprises a VL chain and a first VH chain as described in the previous section, and the second VH chain may comprise HCDRs from the VH region of nesitumumab. The HCDRs defined according to the Kabat nomenclature can have the following sequences. HCDR1: SGDYYWS HCDR2: YIYYSGSTDYNPSLKS HCDR3: VSIFGVGTFDY

[0098] In certain aspects, the second VH chain has the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS It may have an amino acid sequence with at least 80%, at least 90%, at least 95%, or 100% identity thereto.

[0099] The second VH chain of the bispecific antibody that binds to MDR1 and EGFR has the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK It may be present in a heavy chain having an amino acid sequence with at least 80%, at least 90%, at least 95%, or 100% identity thereto.

[0100] In a second aspect, the anti-MDR1 anti-EGFR bispecific antibody comprises a VL chain and a first VH chain as described in the previous section, and the second VH chain may comprise HCDRs derived from the VH region of cetuximab. The HCDRs defined according to the Kabat nomenclature can have the following sequences. HCDR1: NYGVH HCDR2: VIWSGGNTDYNTPFTS HCDR3: ALTYYDYEFAY

[0101] In certain embodiments, the second VH chain has the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA and may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical thereto.

[0102] The second VH chain of the bispecific antibody that binds to MDR1 and EGFR has the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK and may be present in the heavy chain having an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical thereto.

[0103] The above-mentioned anti-MDR1 anti-EGFR bispecific antibody comprises the same combination of VH and VL chains as the above-mentioned anti-MDR1 anti-CD47 bispecific antibody and anti-MDR1 anti-PD-L1 bispecific antibody, and can be referred to as 15D3 HC::MRK16 LC::Necitumumab HC and 15D3 HC::MRK16 LC::Cetuximab HC antibodies.

[0104] Also provided herein are bispecific antibody molecules that include different combinations of a common VL chain containing an antigen-binding site for MDR1 and a first VH chain containing an antigen-binding site for MDR1, but that are not based on MRK16 LC and / or 15D3 HC as described above.

[0105] In certain embodiments, the anti-MDR1 anti-EGFR bispecific antibody can have the following combinations of heavy and light chains: (i) 15D3 HC::Cetuximab HC::15D3 LC; (ii) MRK16 HC::Cetuximab HC:15D3 LC; or (iii) MRK16 HC::Cetuximab HC::MRK16 LC.

[0106] The HCDR and LCDR, VL and VH regions, and heavy and light chains may have the same sequences as provided herein.

[0107] The first HC can include charge substitutions K392D and K409D, and the second heavy chain can include charge substitutions E356K and D399K, or vice versa.

[0108] In one embodiment, the bispecific antibodies of the disclosure do not include the bispecific antibody UIC2 DD HC::Cetuximab KK HC::MRK16 LC. This bispecific antibody is a bispecific antibody that includes a VH chain comprising HCDR 1-3 of the anti-MDR1 antibody UIC2. However, this antibody does not retain binding to MDR1 as measured by FACS using 293T cells overexpressing MDR1. See Figure 31. The VH chain of UIC2 HC is as provided elsewhere herein.

[0109] In certain embodiments, the bispecific antibody comprises a second heavy chain comprising a second VH chain and a heavy chain constant region as described herein. The heavy chain may comprise a human IgG1 heavy chain constant region sequence.

[0110] In certain embodiments, the bispecific antibody molecule specifically binds to cells that express both MDR1 and TAA, and has a greater than 2-fold affinity for cells that express both MDR1 and TAA compared to cells that express either MDR1 or TAA.

[0111] In certain embodiments, the bispecific antibody molecule can increase the sensitivity of cancer cells to treatment with chemotherapeutic agents, where the half maximal inhibitory concentration (IC50) of the chemotherapeutic agent when co-administered with the antibody is at least 2-fold lower (e.g., at least 3-fold lower, at least 4-fold lower, at least 5-fold lower, at least 10-fold lower, at least 20-fold lower, or at least 30-fold lower) than the IC50 of the chemotherapeutic agent when co-administered with an anti-MDR1 antibody comprising a VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA and a VL chain having the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK In certain embodiments, the anti-MDR1 antibody can be the 15D3 antibody described in U.S. Patent No. 5,959,084.

[0112] In certain embodiments, the bispecific antibody molecule has the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA has a VH chain having the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK binds to MDR1 with an affinity that is at least two-fold lower (e.g., at least three-fold lower, at least four-fold lower, at least five-fold lower, at least ten-fold lower, at least twenty-fold lower, or at least thirty-fold lower) than an anti-MDR1 antibody comprising a VL chain having the following sequence:

[0113] In certain embodiments, the bispecific antibody molecule inhibits efflux by MDR1 when bound to a cell expressing MDR1. The inhibition can be a decrease in efflux of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or more as compared to efflux by MDR1 in the absence of the bispecific antibody.

[0114] In certain embodiments, the bispecific antibody molecule comprises an Fc domain that has been modified to reduce or eliminate binding of the antibody to one or more Fcγ receptors. In one embodiment, the IgG1 Fc domain can have one or more of the substitutions L234A, L235A, P329G and N297A / Q / G.

[0115] As described above, the present disclosure provides a multispecific antibody having a domain targeting a cell efflux pump and a domain targeting a cancer-associated antigen. Multispecific antibodies including a multidrug resistance protein 1 (MDR1) binding domain and a leukocyte surface antigen CD47 binding domain are included. The multispecific antibodies of the present disclosure specifically bind to cells expressing both MDR1 and CD47.

[0116] Accordingly, the multispecific antibodies of the present disclosure target both MDR1 and CD47. MDR1, also known as P-glycoprotein 1 (Pgp), is an energy-dependent efflux pump expressed from ATP-binding cassette subfamily B member 1 (ABCB1) that is involved in the reduction of drug accumulation in multidrug-resistant cells. CD47, also known as integrin-associated protein (IAP), is an immunoglobulin superfamily transmembrane protein that binds to membrane integrins and also functions as a receptor for the ligands thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα), and is encoded by the CD47 gene. CD47 ligand binding can result in the inhibition of phagocytosis, and thus, as a target in immunotherapy, masking of the CD47 extracellular domain prevents the inhibition of immune-mediated killing of CD47-expressing cancer cells.

[0117] A schematic diagram of one embodiment of the multispecific antibody of the present disclosure is shown in FIG. 1A. As shown, the multispecific antibody 100 includes all or a portion of an MDR1 binding domain 101, a CD47 binding domain 102, and optionally, an Fc domain 103. As shown in FIG. 1B, in the presence of cells 104 that express MDR1 107 but have low or no CD47 expression, the multispecific antibody 100 has a low affinity for the cells. Similarly, in the presence of cells 105 that express CD47 108 but have low or no MDR1 expression, the multispecific antibody 100 has a low affinity for the cells. However, in the presence of cells 106 that express both MDR1 107 and CD47 108, the multispecific antibody 100 has a high affinity for the cells.

[0118] Accordingly, the multispecific antibodies of the present disclosure bind to cells expressing both MDR1 and CD47 with a higher affinity than cells expressing only MDR1 or CD47. Correspondingly, the multispecific antibodies of the present disclosure bind with a much reduced affinity when each second target is present at a low level compared to, for example, when both the first and second targets are present at levels higher than a low level (e.g., average level, normal level, and / or high level). In some embodiments, the affinity of a subject multispecific antibody for binding to cells expressing both MDR1 and CD47 is greater than two-fold, e.g., greater than 2.5-fold, greater than 3-fold, greater than 4-fold, greater than 5-fold, greater than 6-fold, greater than 7-fold, greater than 8-fold, greater than 9-fold, greater than 10-fold, or more, compared to the affinity of the subject multispecific antibody for binding to cells expressing either MDR1 or CD47 (or either low levels of MDR1 or CD47).

[0119] In some aspects, when a subject multispecific antibody binds to a cell expressing MDR1, it can interfere with the function of the cellular MDR1 protein. Accordingly, the multispecific antibodies of the present disclosure can inhibit efflux by the MDR1 protein, which includes, for example, cases where efflux is reduced by 5% or more, e.g., 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to MDR1-mediated efflux in the absence of the subject multispecific antibody.

[0120] In some embodiments, the multispecific antibody of interest can, when bound to cells expressing CD47, interfere with the function of the cellular CD47 protein. Thus, the multispecific antibodies of the present disclosure can inhibit the binding of a CD47 ligand or CD47 binding partner to CD47, which includes, for example, a reduction in ligand / binding partner binding of 5% or more, for example 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to binding by CD47 in the absence of the multispecific antibody of interest.

[0121] The multispecific antibodies of the present disclosure are at least bispecific for MDR1 and CD47, where the composition of the antibody can vary. The term “antibody” refers to a protein comprising one or more (e.g., one or two) heavy chain variable regions (VH) and / or one or more (e.g., one or two) light chain variable regions (VL), or a fragment thereof that can bind to an epitope. With respect to the multispecific antibodies described herein, such antibodies can bind to at least two different epitopes present on two different target proteins. The number of different target proteins, i.e., the number of different epitopes, bound by the multispecific antibody of interest can vary and can be two (i.e., bispecific), three (trispecific), four, or more.

[0122] In one embodiment, the multispecific antibodies of the present disclosure can include a common light chain. As used herein, the term “common light chain” generally refers to the use and incorporation of two copies of the same light chain in a multispecific antibody. Put another way, in the constructed multispecific antibody, the light chain associates with the MDR1-specific heavy chain and a second copy of that same light chain associates with the CD47-specific heavy chain.

[0123] The VH and VL regions can be further subdivided into regions of hypervariability called "complementary determining regions (CDRs)", with more conserved regions called "framework regions (FRs)" interspersed therebetween. The ranges of the FRs and CDRs are precisely defined (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1987) J. Mol. Biol. 196: 901-917). VH can contain three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, VL can contain three CDRs and four FRs arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0124] The VH or VL chain of an antibody can further include all or part of the heavy or light chain constant region, thereby forming an immunoglobulin heavy or light chain, respectively. In one embodiment, the antibody is a tetramer of two heavy chains and two light chains, where the heavy and light chains are interconnected, for example, by disulfide bonds. The heavy chain constant region is composed of three domains called CH1, CH2, and CH3. The light chain constant region is composed of one domain, CL. The variable regions of the heavy and light chains include the binding regions that interact with the antigen. The constant region of the antibody typically mediates the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system. The term "antibody" includes immunoglobulins of the IgA, IgG, IgE, IgD, IgM types and their subtypes. In certain embodiments, the subject antibody is of the IgG isotype.

[0125] As used herein, the term "immunoglobulin" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes; and numerous immunoglobulin variable region genes. A full-length immunoglobulin light chain (about 25 kD or 214 amino acids) is encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region at the C-terminus. A full-length immunoglobulin heavy chain (about 50 kD or 446 amino acids) is encoded by a variable region gene at the N-terminus (about 116 amino acids) and one of the other constant region genes at the C-terminus, such as gamma (encoding about 330 amino acids). In certain embodiments, the subject antibody comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0126] In certain embodiments, the subject antibody does not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, and instead comprises one or more antigen-binding fragments of a full-length immunoglobulin heavy chain and / or one or more antigen-binding fragments of a full-length immunoglobulin light chain. In some embodiments, these antigen-binding fragments are contained on separate polypeptide chains. In other embodiments, these antigen-binding fragments are contained within a single polypeptide chain.

[0127] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to MDR1 or CD47 as described above. Examples of binding fragments include: (i) Fab fragment (a monovalent fragment consisting of, for example, VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragment (a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region); (iii) Fd fragment (containing, for example, consisting of VH and CH1 domains); (iv) Fv fragment (containing, for example, consisting of the VH and VL domains of a single arm of an antibody); (v) dAb fragment (containing, for example, consisting of a VH domain); (vi) isolated CDR; (vii) single-chain Fv (scFv) (including those in which the VH and VL domains of a single arm of an antibody are connected by a synthetic linker using recombinant means and the VH and VL domains are paired to form a monovalent molecule, for example, consisting of that); (viii) diabody (containing two scFvs in which the VH and VL domains are linked so as not to pair to form a monovalent molecule, for example, consisting of two such scFvs; each VH of the scFv pair forms a pair with the VL domain of the other scFv to form a divalent molecule).

[0128] In certain embodiments, the subject antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized or in vitro generated antibody. As used herein, the term “recombinant” or “modified” antibody is intended to include all antibodies prepared, expressed, made, or isolated by recombinant means, e.g., (i) an antibody expressed using a recombinant expression vector transfected into a host cell; (ii) an antibody isolated from a recombinant combinatorial antibody library; (iii) an antibody isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes; or (iv) an antibody prepared, expressed, made, or isolated by other means including covalently linking human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized antibodies, CDR-grafted antibodies, chimeric antibodies, deimmunized antibodies, and in vitro generated antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences.

[0129] Modified antibodies may contain modified domains, including where any antibody domain may be modified from its naturally occurring form. In certain embodiments, the modified antibody contains a modified CH2 and / or a modified CH3 domain, contains a modified Fc domain, and may contain a modified heavy chain. In some examples, the modified Fc domain may utilize electrostatic steering effects, which include, but are not limited to, those through use of the procedures described in, e.g., Gunasekeran et al, (2010) Journal of Biological Chemistry 285, 19637-19646, the disclosure of which is incorporated herein by reference in its entirety. In some examples, bispecific antibodies are assembled via charge substitution in the CH3 domain, including, but not limited to, those in which one heavy chain is modified to contain K392D and K409D substitutions and the other heavy chain is modified to contain E356K and D399K substitutions. The charge substitution chains preferentially form heterodimers with each other. Amino acid substitutions are numbered according to the EU numbering system for HC.

[0130] In some examples, the antibodies of the present disclosure include charge substitutions. In some examples, the antibodies of the present disclosure do not include charge substitutions. In some examples, alternative means to promote preferential heterodimer formation of the desired chains may be used.

[0131] In some cases, the modified heavy chain may include a knob-into-hole modification. The "knob-into-hole" amino acid modification is a rational design strategy in antibody engineering and is used for heavy chain heterodimerization and the production of multispecific antibodies (including bispecific IgG antibodies). For example, when incorporating the knob-into-hole strategy into a bispecific antibody made from two monoclonal antibodies with different specificities, amino acid changes are engineered to form a "knob" on the CH3 of the heavy chain of monoclonal antibody 1 (mAb1) and a "hole" on the CH3 of the heavy chain of monoclonal antibody 2 (mAb2). The knob can be represented by a large amino acid such as tyrosine (Y), and the hole can be represented by a small amino acid such as threonine (T). For example, the knob-into-hole pair modification can be generated by a T22Y substitution in the first CH3 domain and a Y86T substitution in the partner CH3 domain. Examples of knob-into-hole modifications are described in Carter, J. Immunol. Methods, 248(1-2):7-15 (2001); Ridgway, J. B. et al. Protein Eng. 9(7):617-2 (1996); and Merchant, A. M. et al. Nat. Biotechnol. 16(7):677-81 (1998), the disclosures of which are incorporated herein by reference in their entireties. In antibodies generated from paired knob-into-hole modified domains, the bispecific heterodimer will generally represent the major fraction.

[0132] As summarized above, the multispecific antibodies of the present disclosure include an MDR1 binding domain and a CD47 binding domain. Such domains can vary, including, for example, the epitopes bound by the domain, the arrangement and sequence of the variable regions, and the like.

[0133] The MDR1 binding domain of the present invention specifically binds to one or more epitopes of MDR1. Thus, the epitope is an MDR1 epitope. The size of the MDR1 epitope bound by the MDR1 binding domain can vary, and the MDR1 epitope can be formed by a polypeptide having a continuous stretch of an MDR1 sequence that can range from 4 aa or less to 12 aa or more, including, but not limited to, 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 aa, 5 aa - 8 aa, 6 aa - 8 aa, etc.

[0134] In some embodiments, the MDR1 epitope can be formed by a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an adjacent stretch of an MDR1 sequence, including, but not limited to, for example: Human MDR1 sequence Or a rodent MDR1 sequence, such as the following mouse MDR1 sequence: or a sequence of a non-human primate, such as the following Pan troglodytes (chimpanzee) sequence: or the following Macaca fascicularis (cynomolgus monkey) sequences: etc.

[0135] The MDR1 binding domain of the present invention exhibits high - affinity binding to MDR1. For example, the MDR1 binding domain of the present invention binds to MDR1 with an affinity of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M, or with an affinity greater than 10 -12 M. The MDR1 binding domain of the present invention binds to the epitope present on MDR1 with an affinity of about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, or about 10 -11 M to about 10 -12 M, or with an affinity greater than 10 -12 M.

[0136] The MDR1 binding domain of the present invention does not substantially bind to other proteins that are related but have different sequences, for example, to epitopes formed by amino acids within related but different EPs. The binding of the MDR1 binding domain to epitopes formed by amino acids within related but different proteins is generally non - specific binding with a substantially lower affinity than the specific binding of the MDR1 binding domain to the epitope on MDR1. A substantially lower affinity is generally at least 2 - fold, 3 - fold, 5 - fold, 10 - fold, 50 - fold, 100 - fold, 500 - fold, or 1000 - fold lower.

[0137] The MDR1 binding domain of the present invention can reduce the transport of molecules via the MDR1 transporter. For example, the MDR1 binding domain of the present invention can reduce transport by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more compared to the degree of transport in the absence of the MDR1 binding domain.

[0138] In certain embodiments, the antibodies of the invention comprise: a) i. A CDR1 region that is identical to or comprises the heavy chain CDR1 region of RYTMS in amino acid sequence; ii. A CDR2 region that is identical to or comprises the heavy chain CDR2 region of TISSGGGNTYYPDSVKG in amino acid sequence; and iii. A CDR3 region that is identical to or comprises the heavy chain CDR3 region of YGAGDAWFAY in amino acid sequence comprising a heavy chain variable domain. These CDR 1-3 regions have the following sequences: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA the VH chain of the 15D3 antibody having, or the following sequence: evqlvesggvvvqpggslrlscaasgftfsrytmswvrqapgkglewvatissgggX 2 tyypdsvkgrftvsrdnsknslylqmnslrtedtalyycarygagdawfaywgqgtlvtvss present in a humanized version of this VH chain having, where X 2 is N, Q or S. These CDRs are based on Kabat nomenclature.

[0139] In some embodiments, the subject antibody has a heavy chain variable region comprising one, two, or three heavy chain CDRs selected from one or more of CDR1 (RYTMS), CDR2 (TISSGGGX 2 TYYPDSVKG, where X 2 is N, Q, or S), and CDR3 (YGAGDAWFAY), and an FR region that is a mammalian sequence including, for example, rodent, non-human primate, and human sequences (e.g., those encoded by their respective heavy chain FR coding sequences). For example, in some aspects, the subject antibody comprises, in order from the N-terminus to the C-terminus, human heavy chain FR1, CDR1 comprising the amino acid sequence RYTMS; human heavy chain FR2; CDR2 comprising TISSGGGNTYYPDSVKG; human heavy chain FR3; CDR3 comprising the amino acid sequence YGAGDAWFAY; and human heavy chain FR4.

[0140] The subject antibody can comprise a heavy chain variable region comprising an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including 100%) identity to the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS The antibodies of the invention can comprise one, two, or three heavy chain complementarity determining regions (CDRs) having an amino acid sequence selected from one or more of CDR1 (RYTMS), CDR2 (TISSGGGNTYYPDSVKG), and CDR3 (YGAGDAWFAY). In certain aspects, the VH chain is humanized to include a human framework region, and the humanized VH chain has the following sequence: evqlvesggvvvqpggslrlscaasgftfsrytmswvrqapgkglewvatissgggX 2tyypdsvkgrftvsrdnsknslylqmnslrtedtalyycarygagdawfaywgqgtlvtvs may comprise an amino acid sequence having an identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including 100%) to, where X 2 is N, Q or S.

[0141] The subject CD47 binding domain specifically binds to one or more epitopes of CD47. Thus, the epitope is a CD47 epitope. The size of the CD47 epitope bound by the CD47 binding domain can vary, which includes the case where the CD47 epitope is formed by a polypeptide having a continuous stretch of a CD47 sequence in the range of 4 aa or less to 12 aa or more, including but not limited to, 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 aa, 5 aa - 8 aa, 6 aa - 8 aa, etc.

[0142] In some embodiments, the CD47 epitope can be formed by a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous stretch of a CD47 sequence such as, but not limited to, the following: Human CD47 sequence: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE; or a rodent CD47 sequence, such as the following mouse CD47 sequence: MWPLAAALLLGSCCCGSAQLLFSNVNSIEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTTTDQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSWFSPNEKILIVIFPILAILLFWGKFGILTLKYKSSHTNKRIILLLVAGLVLTVIVVVGAILLIPGEKPVKNASGLGLIVISTGILILLQYNVFMTAFGMTSFTIAILITQVLGYVLALVGLCLCIMACEPVHGPLLISGLGIIALAELLGLVYMKFVASNQRTIQPPRNR; a non-human primate sequence, such as the following Pongo abelii (Sumatran orangutan) sequence: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLIITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLNSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE or Macaca mulatta (rhesus monkey) sequence: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE etc.

[0143] The CD47 binding domain of the present invention exhibits high affinity binding to CD47. For example, the subject CD47 binding domain is at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M, or 10 -12It binds to CD47 with an affinity greater than M. The subject CD47-binding domain binds to an epitope present on CD47 with an affinity of about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, or about 10 -11 M to about 10 -12 M, or 10 -12 It binds with an affinity greater than M.

[0144] The CD47-binding domain of the present invention does not substantially show binding to epitopes formed by amino acids in other proteins that are related but have different sequences, such as immune checkpoint markers that are related but have different sequences. The binding of the subject CD47-binding domain to epitopes formed by amino acids in related but differently sequenced proteins is generally non-specific binding with a substantially lower affinity than the specific binding of the CD47-binding domain to epitopes on CD47. A substantially lower affinity is generally at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower affinity.

[0145] The subject CD47-binding domain can reduce the binding of CD47-binding partners to CD47, including, for example, thrombospondin-1 (TSP-1), signal regulatory protein α (SIRPα), and integrins (e.g., integrin avb3). For example, the subject CD47-binding domain can reduce CD47-binding partner binding by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the degree of binding in the absence of the CD47-binding domain.

[0146] In certain embodiments, the subject antibody comprises a variable domain that includes: a) i. a CDR1 region that is identical to or includes the NYNMH heavy chain CDR1 region in amino acid sequence; ii. a CDR2 region that is identical to or includes the TIYPGNDDTSYNQKFKD heavy chain CDR2 region in amino acid sequence; iii. a CDR3 region that is identical to or includes the GGYRAMDY heavy chain CDR3 region in amino acid sequence. These CDR 1-3 regions have the following sequences: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS and are present in the VH chain of the 5F9 antibody having the following sequence. These CDRs are based on the Kabat nomenclature.

[0147] The subject antibody can include a heavy chain variable region that includes an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including 100%) identity to the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS The antibody of the invention can include a heavy chain variable region that includes one, two, or three heavy chain complementarity determining regions (CDRs) having an amino acid sequence selected from one or more of CDR1 (NYNMH), CDR2 (TIYPGNDDTSYNQKFKD), and CDR3 (GGYRAMDY).

[0148] In some embodiments, the subject antibody comprises one, two, or three heavy chain CDRs having an amino acid sequence selected from one or more of CDR1 (NYNMH), CDR2 (TIYPGNDDTSYNQKFKD), and CDR3 (GGYRAMDY), and a FR region that is a mammalian, e.g., rodent, non-human primate, or human sequence (e.g., encoded by the respective heavy chain FR-encoding sequences). For example, in some aspects, the subject antibody comprises, in order from the N-terminus to the C-terminus, a human heavy chain FR1; CDR1 comprising the amino acid sequence NYNMH; a human heavy chain FR2; CDR2 comprising the amino acid sequence TIYPGNDDTSYNQKFKD; a human heavy chain FR3; CDR3 comprising the amino acid sequence GGYRAMDY; and a heavy chain variable region comprising a human heavy chain FR4.

[0149] The subject antibody has the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK or a humanized version of this sequence having the following sequence: divmtqtplsspvtlgqpasiscrssqsivhstgX 1 tylewyqqrpgqpprlliykisnrfsgvpdrfsgsgagtdftlkisrveaedvgvyycfqashfprtfgggtkleik and can comprise a light chain variable region having an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more (including 100%) identical to the sequence: 1 where X is N, Q, or S.

[0150] The subject antibody has CDR1 (RSSQSIVHSTG X 1 TYLEW; where X 1It can include a light chain variable region that includes light chain CDRs having the amino acid sequences represented by CDR1 (RSSQSLLHSDGFDYLNW), CDR2 (KISNRFSG), and CDR3 (FQASHFPRTF), or CDR1 (RSSQSLLHSDGFDYLNW), CDR2 (ALSNRASG), and CDR3 (MZALQAPITF). In some examples, such light chain variable regions can be used in the common light chain of a multispecific antibody of interest.

[0151] In some embodiments, the antibody of interest has a light chain variable region that includes light chain CDRs having the amino acid sequences represented by CDR1 (RSSQSIVHSTG X 1 TYLEW; where X 1 is N, Q, or S), CDR2 (KISNRFSG), and CDR3 (FQASHFPRTF), or CDR1 (RSSQSLLHSDGFDYLNW), CDR2 (ALSNRASG), and CDR3 (MZALQAPITF), and an FR region that is a mammalian, e.g., rodent, non-human primate, or human sequence (e.g., encoded by the respective light chain FR-encoding sequences). For example, in some aspects, the antibody of interest has, in order from the N-terminus to the C-terminus, human light chain FR1; CDR1 that includes the amino acid sequence RSSQSIVHSTG X 1 TYLEW (where X 1 is N, Q, or S); human light chain FR2; CDR2 that includes the amino acid sequence KISNRFSG; human light chain FR3; CDR3 that includes the amino acid sequence represented by FQASHFPRTF; and human light chain FR4.

[0152] In some embodiments, the subject antibody optionally comprises a heavy chain FR1 region; a CDR1 comprising the amino acid sequence RYTMS; a heavy chain FR2 region; a CDR2 comprising the amino acid sequence TISSGGG(N / S / Q)TYYPDSVKG; a heavy chain FR3 region; a CDR3 comprising the amino acid sequence YGAGDAWFAY; and a heavy chain FR4 region; and optionally a heavy chain FR1 region; a CDR1 comprising the amino acid sequence NYNMH; a heavy chain FR2 region; a CDR2 comprising the amino acid sequence TIYPGNDDTSYNQKFKD; a heavy chain FR3 region; a CDR3 comprising the amino acid sequence GGYRAMDY; and a heavy chain FR4 region. In some of these embodiments, each of the FR regions is a mammalian FR region, including, for example, a human FR region.

[0153] In some embodiments, the subject antibody comprises: the heavy chain sequence described in the preceding paragraph, and further: a light chain FR1 region; a CDR1 comprising the amino acid sequence RSSQSIVHSTGNTYLEW or RSSQSLLHSDGFDYLNW; a light chain FR2 region; a CDR2 comprising the amino acid sequence KISNRFSG or ALSNRASG; a light chain FR3 region; a CDR3 comprising the amino acid sequence FQASHFPRTF or MZALQAPITF; and optionally a light chain FR4 region.

[0154] In one embodiment, the subject antibody has the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG An anti-MDR1 heavy chain sequence comprising a charge-charge swap (KK) modification having: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG An anti-CD47 heavy chain sequence comprising a charge-charge swap (DD) modification having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC And an anti-MDR1 light chain sequence having the following sequence: In some examples, the subject antibody may include an alternative heterodimeric Fc pairing strategy.

[0155] The charge-charge swap modification refers to a substitution in which one heavy chain is modified to include K392D and K409D substitutions and the other heavy chain is modified to include E356K and D399K substitutions. The charge-substituted chains preferably form heterodimers with each other. The numbering of the amino acid substitutions is according to the EU numbering system for Ig HC.

[0156] [Monospecific bivalent antibodies and bispecific antibodies] Also provided herein are antibodies that can be monospecific bivalent antibodies or bispecific antibodies derived therefrom.

[0157] In certain embodiments, the antibodies of the present disclosure have the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (where X 1 Is N, Q or S) and a variable light (VL) chain comprising a light chain CDR (LCDR) of the VL chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Here, X 2 is N, Q, or S) and a variable heavy (VH) chain comprising a heavy chain CDR (HCDR) of the VH chain.

[0158] In certain embodiments, the VL chain has the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK and comprises the LCDR of the VL chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS or the VL chain has the following sequence:[[ID=^{19}]] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK and comprises the LCDR of the VL chain having the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS or the VL chain has the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK comprising the LCDR of the VL chain having the following, and the VH chain has the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS comprising the LCDR of the VH chain having the following.

[0159] In certain embodiments, LCDR1 comprises the sequence RSSQSIVHSTGNTYLE, RSSQSIVHSTGQTYLE, or RSSQSIVHSTGSTYLE; LCDR2 comprises the sequence KISNRFS; and LCDR3 comprises the sequence FQASHFPRT.

[0160] In certain embodiments, HCDR1 comprises the sequence RYTMS; HCDR2 comprises the sequence TISSGGGNTYYPDSVKG, TISSGGGQTYYPDSVKG, or TISSGGGSTYYPDSVKG; and HCDR3 comprises the sequence YGAGDAWFAY.

[0161] In certain embodiments, LCDR1 comprises the sequence RSSQSIVHSTGNTYLE; LCDR2 comprises the sequence KISNRFS; LCDR3 comprises the sequence FQASHFPRT; HCDR1 comprises the sequence RYTMS; HCDR2 comprises the sequence TISSGGGNTYYPDSVKG; and HCDR3 comprises the sequence YGAGDAWFAY.

[0162] In certain embodiments, LCDR1 comprises the sequence RSSQSIVHSTGQTYLE; LCDR2 comprises the sequence KISNRFS; LCDR3 comprises the sequence FQASHFPRT; HCDR1 comprises the sequence RYTMS; HCDR2 comprises the sequence TISSGGGQTYYPDSVKG; and HCDR3 comprises the sequence YGAGDAWFAY.

[0163] In one aspect, LCDR1 comprises the sequence RSSQSIVHSTGSTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGSTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY.

[0164] In one aspect, LCDR1 comprises the sequence RSSQSIVHSTGNTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGQTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY, or LCDR1 comprises the sequence RSSQSIVHSTGNTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGSTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY.

[0165] In one embodiment, LCDR1 comprises the sequence RSSQSIVHSTGQTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGNTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY.

[0166] In one aspect, LCDR1 comprises the sequence RSSQSIVHSTGSTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGNTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY.

[0167] In one aspect, LCDR1 comprises the sequence RSSQSIVHSTGSTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGQTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY.

[0168] In one aspect, LCDR1 comprises the sequence RSSQSIVHSTGNTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGQTYYPDSVKG, and HCDR3 comprises the sequence YGAGDAWFAY.

[0169] In certain aspects, the antibody is a monospecific bivalent antibody that specifically binds to MDR-1. In certain aspects, the monospecific bivalent antibody that specifically binds to MDR-1 comprises a humanized VL chain having an amino acid sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical) to the following amino acid sequence of the VL chain: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK; DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; or DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK.

[0170] In certain embodiments, amino acid residues different from the VL or VH chain sequences are located in the framework regions.

[0171] In certain embodiments, a monospecific bivalent antibody that specifically binds to MDR-1 comprises a humanized VH chain having an amino acid sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical) to the VH chain having the following amino acid sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0172] In certain embodiments, the antibody is a bispecific antibody that comprises a VL chain as a common light chain.

[0173] In certain embodiments, the bispecific antibody comprises an MDR-1 binding domain and a tumor associated antigen (TAA) binding domain, wherein each of the MDR-1 binding domain and the TAA binding domain comprises the LCDR 1-3 of the VL chain.

[0174] In certain embodiments, the TAA is CD47 and the TAA binding domain has the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS comprises the HCDR of the VH chain having

[0175] In certain embodiments, the VH chain of the CD47 binding domain comprises HCDR1 comprising the sequence NYNMH, HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD, and HCDR3 comprising the sequence GGYRAMDY.

[0176] In certain embodiments, the antibody of the present disclosure has the following sequences: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (where X 1 is N, Q or S) and a variable light (VL) chain comprising the light chain CDR (LCDR) of the VL chain, and the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS and a variable heavy (VH) chain comprising the heavy chain CDR (HCDR) of the VH chain having

[0177] In certain embodiments, the VH chain comprises HCDR1 comprising the sequence NYNMH, HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD, and HCDR3 comprising the sequence GGYRAMDY.

[0178] In certain embodiments, the antibody is a monospecific bivalent antibody that specifically binds to CD47. In other embodiments, the antibody is a bispecific antibody that binds to CD47 and MRD-1.

[0179] In certain embodiments, the monospecific bivalent antibodies that specifically bind to MDR-1 and the monospecific bivalent antibodies that specifically bind to CD47, as described herein, can be used in a method for treating cancer in a subject, the method can include co-administering the two antibodies to the subject in an effective amount for treating cancer. In certain embodiments, the method can further include administering a chemotherapeutic agent to the subject.

[0180] The regions and / or chains of the subject antibodies may or may not be linked by one or more linker regions. When present, the linker regions can be from about 5 amino acids to about 50 amino acids in length, for example, about 5 aa to about 10 aa, about 10 aa to about 15 aa, 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 aa to about 45 aa, or about 45 aa to about 50 aa in length.

[0181] Linkers suitable for use in the subject antibodies include "flexible linkers". When present, the linker molecule is generally of sufficient length to allow some flexible movement between the linked regions. The linker molecule is generally about 6 to 50 atoms in length. The linker molecule can be, for example, arylacetylene, an ethylene glycol oligomer containing 2 to 10 monomer units, a diamine, diacids, an amino acid, or a combination thereof. Other linker molecules that can be attached to the polypeptide can be used in light of the present disclosure.

[0182] Suitable linkers can be readily selected and can be of any suitable length among various lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, for example, from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0183] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , GSGGS n (SEQ ID NO: / / ) and GGGS n (SEQ ID NO: / / ), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and can thus act as a neutral tether between components. Glycine polymers are of particular interest because glycine accesses the phi-psi space even more significantly than even alanine and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO: / / ), GGSGG (SEQ ID NO: / / ), GSGSG (SEQ ID NO: / / ), GSGGG (SEQ ID NO: / / ), GGGSG (SEQ ID NO: / / ), GSSSG (SEQ ID NO: / / ), etc. One of ordinary skill in the art will recognize that the design of a peptide linked to any of the above elements can include a linker that is all or partially flexible, and thus the linker can include a flexible linker and one or more portions that impart a less flexible structure.

[0184] In one aspect, the subject antibody is "humanized." The term "humanized antibody" refers to an antibody that includes at least one chain that includes variable region framework residues substantially derived from human antibody chains (referred to as acceptor immunoglobulins or acceptor antibodies) and at least one CDR substantially derived from a non-human antibody (e.g., a rodent (e.g., a mouse antibody), a non-human primate, etc.) (referred to as a donor immunoglobulin or donor antibody). See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761, WO 90 / 07861, and U.S. Pat. No. 5,225,539. The constant region(s), if present, may be substantially or completely derived from human immunoglobulins. In some embodiments, the subject antibody includes one or more MDR1 CDRs, and one or more CD47 CDRs, and one or more FR regions from a human antibody. Methods of making humanized antibodies are known in the art. See, for example, U.S. Patent No. 7,256,273.

[0185] Replacing mouse CDRs into the human variable domain framework can result in the maintenance of their correct spatial orientation, where, for example, the human variable domain framework adopts the same or a similar conformation as the mouse variable domain framework from which its CDR is derived. This can be achieved by obtaining human variable domains from human antibodies having framework sequences that exhibit a high degree of sequence identity with the mouse variable framework domain from which the CDR is derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

[0186] Once the complementarity determining regions of the mouse donor immunoglobulin and the appropriate human acceptor immunoglobulin have been identified, the next step is to determine whether and which residues should be replaced from these components in order to optimize the properties of the resulting humanized antibody. Generally, the introduction of mouse residues should be minimized since it increases the risk that the antibody will induce a human anti-mouse antibody (HAMA) response in humans. Methods recognized in the art for measuring immune responses can be performed to monitor HAMA responses in a particular patient or during clinical trials. Patients administered the humanized antibody can be subject to immunogenicity evaluations at the start of the treatment and during the administration period. The HAMA response is measured by detecting antibodies against the humanized therapeutic reagent in serum samples from the patient using methods known to those of skill in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid phase ELISA assays. In many embodiments, the subject humanized antibody does not substantially induce a HAMA response in a human subject.

[0187] Particular amino acids from human variable region framework residues are selected for substitution based on their possible effects on the conformation of the CDR and / or its binding to antigen. The unnatural juxtaposition of mouse CDR regions and human variable framework regions can result in unnatural conformational constraints, which lead to a loss of binding affinity unless corrected by substitution of specific amino acid residues.

[0188] The selection of amino acid residues for substitution can be determined, at least in part, by computer modeling. Computer hardware and software for creating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are created starting from the solved structure of an immunoglobulin chain or its domain. The amino acid sequence similarity between the chain to be modeled and the chain or domain of the solved three-dimensional structure is compared, and the chain or domain with the greatest sequence similarity is selected as the starting point for molecular model construction. Chains or domains sharing at least 50% sequence identity are selected for modeling, preferably those sharing at least 60%, 70%, 80%, 90% sequence identity or more. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain to be modeled and the amino acids in the starting structure. The modified structure is assembled into a complex immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are at appropriate distances from each other and that bond lengths and angles are within chemically acceptable ranges.

[0189] The CDR and framework regions are as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). Alternative structural definitions have been proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol. 186:651 (1989) (collectively referred to as "Chothia"). When the framework residues defined by Kabat above constitute the structural loop residues defined by Chothia above, the amino acids present in the mouse antibody can be selected for substitution into the humanized antibody. "Residues adjacent to the CDR region" include the amino acid residues at positions directly adjacent to one or more of the CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., positions directly adjacent to the CDRs defined by Kabat or the CDRs defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with the amino acids in the CDR, and if they are selected from the acceptor, they are likely to distort the donor CDR and reduce affinity. Furthermore, adjacent amino acids can interact directly with the antigen (Amit et al., Science, 233:747 (1986)), and it may be desirable to select these amino acids from the donor to maintain all antigen contacts that provide affinity in the original antibody.

[0190] In one aspect, the subject antibody comprises an scFv multimer. For example, in some embodiments, the subject antibody is an scFv dimer (e.g., one containing two tandem scFvs (scFv2)), an scFv trimer (e.g., one containing three tandem scFvs (scFv3)), an scFv tetramer (e.g., one containing four tandem scFvs (scFv4)), or a multimer of more than four scFvs (e.g., in tandem). The scFv monomers can be linked in tandem via a linker having a length of about 2 amino acids to about 10 amino acids, e.g., a linker having a length of 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. Suitable linkers include, for example, (Gly) x where x is an integer from 2 to 10. Other suitable linkers are as described above. In some embodiments, each of the scFv monomers in the subject scFV multimer is humanized as described above.

[0191] In one aspect, the subject antibody comprises a constant region of an immunoglobulin (e.g., an Fc region). The Fc region, when present, can be a human Fc region. When a constant region is present, the antibody can comprise constant regions of both the light and heavy chains. Suitable heavy chain constant regions include the CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include antibodies having constant regions of all types including IgM, IgG, IgD, IgA and IgE, and all isotypes including IgG1, IgG2, IgG3 and IgG4. An example of a suitable heavy chain Fc region is the human isotype IgG1 Fc. The light chain constant region can be lambda or kappa. The subject antibody (e.g., the subject humanized antibody) can comprise sequences from multiple classes or isotypes. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy chains, as light chains, as Fab, Fab' F(ab')2, and Fv, or as a single chain antibody in which the heavy and light chain variable domains are linked via a spacer.

[0192] In some embodiments, the target antibody contains a free thiol (-SH) group at its carboxyl terminus, and this free thiol group can be used to bind the antibody to a second polypeptide (e.g., another antibody including the antibody of the present invention), a scaffold, a carrier, etc.

[0193] The target antibody can be covalently bound to a second moiety (e.g., a lipid, a polypeptide other than the target antibody, a synthetic polymer, a carbohydrate, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks polypeptides via its amino moiety. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be crosslinked. Homobifunctional NHS esters and imidoesters crosslink amine-containing polypeptides. At a weakly alkaline pH, imidoesters react only with primary amines to form imidoamides, and the overall charge of the crosslinked polypeptides is not affected. Homobifunctional sulfhydryl-reactive crosslinkers include bis maleimide hexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propionamide butane (DPDPB).

[0194] [Compositions and Formulations] The present disclosure provides a composition comprising a target antibody. The target antibody composition may, in addition to the target antibody, include one or more of salts such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizing agents; detergents such as nonionic detergents like Tween-20; protease inhibitors; glycerol; and the like.

[0195] The compositions of the present disclosure also include pharmaceutical compositions comprising the multispecific antibodies described herein. Generally, the formulation contains an effective amount of the target antibody. "Effective amount" means a dosage sufficient to produce a desired result, such as a reduction in cancer in a subject, a decrease in the growth rate of cancer in a subject, an improvement in cancer symptoms, etc. Generally, the desired result is at least a reduction in cancer symptoms, a decrease in cancer growth, a decrease in cancer size, etc., compared to a control. The target antibody can be delivered or formulated in a manner that bypasses the blood-brain barrier. In some examples, the antibody can include a delivery enhancer, which can facilitate, for example, passage through the blood-brain barrier, increased permeability, such as efficient transdermal delivery, when such an enhancer enables this. Useful delivery enhancers include, but are not limited to, for example, ceroport, legadenosine, borneol, puerarin, propylene glycol, oleic acid, azone, N-methylpyrrolidone, Tween 80, limonene, lipid-based nanoparticles (NP), liposomes, niosomes, transferosomes, ethosomes, dendrimers, micelle NPs, polymeric nanostructures, metallic nanostructures, magnetic nanostructures, recombinant human hyaluronidase, and the like.

[0196] In this method, the present antibody can be administered to a host using any convenient means that can bring about the desired therapeutic or diagnostic effect. Thus, the agent can be incorporated into various formulations for therapeutic administration. More specifically, the subject antibody can be formulated into a pharmaceutical composition in combination with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated in solid, semi-solid, liquid or gaseous form, for example, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosol preparations.

[0197] In a pharmaceutical dosage form, the subject antibody can be administered together with pharmaceutically acceptable excipients, or they can be used alone, or in appropriate association with and combination with other pharmaceutically active compounds. The following methods and excipients are merely illustrative and in no way limiting.

[0198] For oral formulations, the subject antibody can be used alone or in combination with suitable additives, such as with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin; with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffers, wetting agents, preservatives and flavoring agents, to produce tablets, powders, granules or capsules. In some examples, oral delivery of the antibody can be enhanced through complexation of the antibody with a suitable hydrogel.

[0199] The subject antibody can be formulated into an injectable preparation by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, and, if desired, with conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents and preservatives.

[0200] A pharmaceutical composition containing the target antibody is prepared by mixing an antibody having a desired purity with any physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer, and / or tonicity agent. Acceptable carriers, excipients, and / or stabilizers are non-toxic to the recipient at the dosages and concentrations used, and buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin and serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronic, Triton-X, polyethylene glycol (PEG).

[0201] The pharmaceutical composition may be in liquid form, lyophilized form, or a liquid form reconstituted from the lyophilized form, where the lyophilized formulation is reconstituted with a sterile solution prior to administration. Standard procedures for reconstituting the lyophilized composition involve adding back a certain volume of pure water (typically equal to the volume removed during lyophilization). However, solutions containing antibacterial agents may be used for the manufacture of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0202] Exemplary antibody concentrations in the pharmaceutical composition can range from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0203] The aqueous formulation of the antibody can be prepared at a pH in a buffer, for example, in the range of about 4.0 to about 7.0, about 5.0 to about 6.0, or about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the desired tonicity of the buffer and the formulation.

[0204] A tonicity agent can be included in the antibody formulation to adjust the tonicity of the formulation. Typical tonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions can also be appropriate. The term "isotonic" means a solution having the same tonicity as another solution (e.g., physiological saline or serum) to which it is compared. The tonicity agent can be used in an amount from about 5 mM to about 350 mM, for example, from 100 mM to 350 nM.

[0205] Surfactants can also be added to the antibody formulation to reduce aggregation of the formulated antibody and / or minimize particle formation and / or reduce adsorption in the formulation. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trade name Tween 20) and polysorbate 80 (sold under the trade name Tween 80). Examples of suitable polyethylene-polypropylene copolymers are those sold under the name Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trade name Brij™. Exemplary concentrations of the surfactant can range from about 0.001% to about 1% w / v.

[0206] To protect labile active ingredients (e.g., proteins) from destabilizing conditions during the lyophilization process, a lyoprotectant may be added. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant can be included in an amount from about 10 mM to 500 nM.

[0207] In one aspect, the subject formulation comprises the subject antibody and one or more of the above-identified substances (e.g., surfactant, buffer, stabilizer, tonicity agent), and is essentially free of one or more preservatives such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other aspects, the preservative is included in the formulation at a concentration, for example, in the range of about 0.001% to about 2% (w / v).

[0208] For example, the subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration, and can include from about 1 mg / mL to about 200 mg / mL of the subject antibody; from about 0.001% to about 1% of at least one surfactant; from about 1 mM to about 100 mM of a buffer; optionally from about 10 mM to about 500 mM of a stabilizer; and from about 5 mM to about 305 mM of a tonicity agent, and has a pH from about 4.0 to about 7.0.

[0209] As another example, the subject parenteral formulation is a liquid or lyophilized formulation that includes from about 1 mg / mL to about 200 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, and has a pH of 5.5.

[0210] As another example, the subject parenteral formulation includes a lyophilized formulation comprising: 1) 15 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, pH 5.5; or 2) 75 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, pH 5.5; or 3) 75 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; pH 5.5; or 4) 75 mg / mL of the subject antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose, pH 5.5; or 6) 75 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose, pH 5.5

[0211] As another example, the subject parenteral formulation is a liquid formulation comprising: 1) 7.5 mg / mL of the subject antibody; 0.022% Tween 20 w / v; 120 mM L-histidine; and 250 125 mM sucrose, pH 5.5; or 2) 37.5 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose, pH 5.5; or 3) 37.5 mg / mL of the subject antibody; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose, pH 5.5; or 4) 37.5 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 10 mM L-histidine; and 125 mM trehalose, pH 5.5; or 5) 37.5 mg / mL of the subject antibody; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM trehalose, pH 5.5; or 6) 5 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose, pH 5.5; or 7) 75 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol, pH 5.5; or 8) 75 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride, pH 5.5; or 9) 150 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; 250 mM trehalose; pH 5.5; or 10) 150 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol, pH 5.5; or 11) 150 mg / mL of the subject antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride, pH 5.5; or 12) 10 mg / mL of the subject antibody; 0.01% Tween 20 w / v; 20 mM L-histidine; and 40 mM sodium chloride, pH 5.5.

[0212] The antibodies of the present invention can be used in aerosol formulations for administration by inhalation. The subject antibodies can be formulated with a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, etc.

[0213] Furthermore, the antibodies of the present invention can be made into suppositories by mixing with various substrates such as an emulsified substrate or a water-soluble substrate. The subject antibodies can be administered rectally via suppositories. The suppository may contain a vehicle that melts at body temperature but solidifies at room temperature, such as cocoa butter, carbowax, and polyethylene glycol.

[0214] Unit dosage forms for oral or rectal administration can be provided, such as syrups, elixirs, and suspensions, where each dosage unit, e.g., one cup of tea, one tablespoon, tablet, or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration can contain the subject antibody in the composition as a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier.

[0215] As used herein, the term "unit dosage form" refers to physically discrete units suitable as a single dosage for human and animal subjects, and each unit contains a predetermined amount of the compound of the present invention calculated to produce the desired effect, accompanied by a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of the subject antibodies can depend on the particular antibody used, the effect to be achieved, and the pharmacokinetics associated with each antibody in the host.

[0216] Other modes of administration can also be found in use with the present invention. For example, the subject antibodies can be formulated into suppositories and, in some cases, into aerosols and intranasal compositions. For suppositories, the vehicle composition includes conventional binders and carriers such as polyalkylene glycols or triglycerides. Such suppositories can be formed from a mixture containing an active ingredient in the range of about 0.5% to about 10% (w / w), e.g., about 1% to about 2%.

[0217] Nasal preparations usually contain a vehicle that causes no irritation to the nasal mucosa and does not significantly interfere with the ciliary function. Diluents such as water, physiological saline or other known substances can be used with the present invention. Nasal preparations may also contain preservatives such as chlorobutanol and benzalkonium chloride, but are not limited thereto. Surfactants may be present to enhance the absorption of the target protein by the nasal mucosa.

[0218] The target antibody can be administered as an injectable preparation. Typically, an injectable composition is prepared as a liquid solution or suspension; it is also possible to prepare a solid form suitable for dissolving or suspending in a liquid vehicle before injection. The preparation may also be emulsified or the antibody may be encapsulated in a liposome carrier.

[0219] Suitable excipient vehicles are, for example, water, physiological saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Further, if desired, the vehicle may contain small amounts of auxiliary substances such as wetting agents or emulsifying agents or pH buffering agents. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or preparation to be administered, in any case, contains an amount of the target antibody sufficient to achieve the desired condition in the subject being treated.

[0220] Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents are generally readily available. Further, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc. are also generally readily available.

[0221] In some embodiments, the subject antibody is formulated into a controlled release formulation. Sustained release preparations can be prepared using methods well known in the art. Suitable examples of sustained release preparations include those in which a semipermeable matrix of a solid hydrophobic polymer contains the antibody, where the matrix is in the form of a shaped article, such as a film or a microcapsule. Examples of sustained release matrices include polyesters, copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. The possibility of loss of biological activity and changes in immunogenicity of the antibody contained in the sustained release preparation can be prevented by using appropriate additives, controlling the water content, and developing specific polymer matrix compositions.

[0222] Controlled release within the scope of the present invention can be construed to mean any of a number of extended release dosage forms. The following terms may be considered to be substantially equivalent to controlled release for the purposes of the present invention: continuous release, controlled release, delayed release, depot, sustained release, long-term release, programmed release, extended release, proportional release, retarded release, persistence, delay, slow release, spaced release, sustained release, time coat, timed release, delayed action, extended action, stratified time action, long-lasting action, extended action, repeated action, slow action, sustained action drug, and extended release. Further discussion of these terms is described in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0223] [Dosage and Administration] The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage for an individual patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, the patient's gender, time and route of administration, general health status, and other drugs being administered simultaneously. The subject antibody can be administered in an amount between 1 ng / kg body weight to 20 mg / kg body weight per administration, such as 0.1 mg / kg body weight to 10 mg / kg body weight, such as 0.5 mg / kg body weight to 5 mg / kg body weight; however, particularly considering the aforementioned factors, dosages below or above this exemplary range are also contemplated. If the regimen is a continuous infusion, it may be in the range of 1 μg to 10 mg / kg body weight per minute.

[0224] One of ordinary skill in the art will readily understand that the dosage level can vary as a function of the specific antibody, the severity of the symptoms, and the subject's sensitivity to side effects. The preferred dosage of a given compound can be readily determined by one of ordinary skill in the art by various means.

[0225] [Route of Administration] The subject antibody is administered to the individual using any available method and route suitable for drug delivery, which includes, for example, in vivo and ex vivo methods, as well as systemic and topical routes of administration.

[0226] 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 of administration. The routes of administration may be combined if desired or may be adjusted according to the antibody and / or the desired effect. The subject antibody composition may be administered as a single dose or multiple doses. In certain embodiments, the subject antibody composition is administered orally. In certain embodiments, the subject antibody composition is administered via an inhalation route. In certain embodiments, the subject antibody composition is administered intranasally. In certain embodiments, the subject antibody composition is administered topically. In certain embodiments, the subject antibody composition is administered intracranially. In certain embodiments, the subject antibody composition is administered intravenously.

[0227] The agent can be administered to the host using any available conventional methods and routes suitable for delivery of conventional agents, including systemic or local routes. Generally, the routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0228] Parenteral routes of administration other than inhalation include, but are not necessarily limited to, local, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than via the gastrointestinal tract. Parenteral administration can be performed to effect systemic or local delivery of the subject antibody. When systemic delivery is desired, administration typically includes local or mucosal administration of a pharmaceutical preparation that is invasive or systemically absorbed.

[0229] The subject antibody can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using suppositories) delivery.

[0230] Treatment means at least an improvement in symptoms associated with a pathological condition that afflicts a host, where improvement is used broadly to denote at least a decrease in the magnitude of a parameter (such as a symptom) associated with the pathological condition being treated, such as cancer and / or cancer growth and associated pain. As such, treatment includes situations where the pathological condition or at least the symptoms characterizing it are completely suppressed, such that the host no longer suffers from the pathological condition or at least the symptoms, e.g., is prevented from occurring or is stopped, e.g., terminated.

[0231] Various hosts (where the term "host" is used interchangeably herein with the terms "subject", "individual", and "patient") are treatable according to the methods of the invention. Generally, such hosts are "mammals" or "mammalian", where these terms are used broadly to describe organisms belonging to the class Mammalia, including the order Carnivora (e.g., dogs and cats), the order Rodentia (e.g., mice, guinea pigs, rats), and the order Primates (e.g., humans, chimpanzees, and monkeys). In one aspect, the host is a human.

[0232] Kits are provided having a unit dose (e.g., an oral or injection dose) of a subject antibody. In some aspects, in addition to a container containing the unit dose, there is an information sheet describing the use of the antibody in treating a pathological condition of interest and the attendant advantages.

[0233] [Nucleic acid] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a subject antibody. The nucleotide sequence encoding the subject antibody can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that enable expression of the nucleotide sequence in an intended target cell (e.g., a cell genetically engineered to synthesize and / or secrete the encoded antibody).

[0234] Suitable promoter and enhancer elements are known in the art. Suitable promoters for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, promoters and enhancer elements of light chain and / or heavy chain immunoglobulin genes; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in the long terminal repeats of retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0235] The nucleotide sequence encoding the subject antibody can be present in an expression vector and / or a cloning vector. If the subject antibody comprises two or more distinct polypeptides, the nucleotide sequences encoding those two polypeptides can be cloned into the same or separate vectors. The distinct polypeptides can be expressed from a single nucleic acid or a single vector using various strategies, such as separate promoters, one or more internal ribosome entry sites (IRES), one or more self-cleaving sequences (e.g., 2A cleavage sequences, e.g., P2A, T2A, E2A, and F2A), combinations thereof, and the like. The expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector.

[0236] A number of suitable vectors and promoters are known to those of ordinary skill in the art; many of them are commercially available for making the recombinant constructs of interest. The following vectors are provided as examples. Bacteria: pbs, phage script, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0237] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker that functions in the expression host may also be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus-based viral vectors (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822 3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613 10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., see J Virol 73:7812-7816, 1999; retroviral vectors (e.g., vectors derived from retroviruses such as murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc. are included.

[0238] As described above, the subject nucleic acid comprises a nucleotide sequence encoding a multispecific antibody of interest. The subject nucleic acid can comprise nucleotide sequences encoding heavy and light chain CDRs, including MDR1 CDR and CD47 CDR. In some embodiments, the subject nucleic acid comprises a nucleotide sequence encoding a heavy chain and / or light chain MDR1 CDR, wherein the sequence encoding the CDR is interspersed with a nucleotide sequence encoding a FR. In some embodiments, the subject nucleic acid comprises a nucleotide sequence encoding a heavy chain and / or light chain CD47 CDR, wherein the sequence encoding the CDR is interspersed with a nucleotide sequence encoding a FR. In one aspect, the nucleotide sequence encoding the FR is a nucleotide sequence encoding a human FR.

[0239] In one aspect, the subject nucleic acid comprises a nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS In one aspect, the subject nucleic acid comprises a nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the following amino acid sequence:

[0240] In one aspect, the subject nucleic acid comprises a nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to.

[0241] In certain embodiments, the subject nucleic acid comprises a nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the following amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to.

[0242] For example, nucleic acids such as those described herein can, in some instances, be introduced into cells, such as by contacting the cells with the nucleic acid. Cells having the introduced nucleic acid are generally referred to herein as genetically modified cells. A variety of methods of nucleic acid delivery can be used, including, but not limited to, for example, naked nucleic acid delivery, viral delivery, chemical transfection, gene gun methods, and the like.

[0243] [Cell] The present disclosure provides isolated genetically recombinant cells (e.g., in vitro cells, ex vivo cells, cultured cells, etc.) that have been genetically modified with a target nucleic acid. In certain embodiments, the isolated genetically recombinant cells can produce a target antibody. Optionally, the genetically recombinant cells can deliver the antibody, for example, to a subject in need thereof. Optionally, the genetically recombinant cells can be used in the production, screening, and / or discovery of bispecific antibodies. The genetically recombinant cells can also, in some instances, include cells in which endogenous gene expression is reduced (e.g., suppressed, knocked down, etc., or eliminated, e.g., knocked out). The genetically recombinant cells can also, in some instances, include cells in which gene expression is enhanced, e.g., cells in which endogenous gene expression or heterologous gene expression is increased.

[0244] Suitable cells include eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as bacterial cells. Introduction of the target nucleic acid into the host cell can be accomplished, for example, by calcium phosphate precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0245] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL 9618, CCL 61, CRL 9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL 1721), COS cells, COS-7 cells (ATCC No. CRL 1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL 1573), HLHepG2 cells, etc., but are not limited thereto.

[0246] In some examples, useful mammalian cells can include cells derived from mammalian tissues or organs. In some examples, the cells used are kidney cells, including kidney cells of an established kidney cell line such as HEK 293T cells, for example.

[0247] Suitable yeast cells or fungal or algal cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, etc.

[0248] Suitable prokaryotic cells include, but are not limited to, any of the laboratory strains such as Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., etc. See, for example, Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that can be used in the present invention include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, laboratory strains are non-pathogenic strains. Other non-limiting examples of suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., etc. In one embodiment, the host cell is Escherichia coli.

[0249] In some examples, the cells of the present disclosure can be immune cells. As used herein, the term "immune cell" generally includes leukocytes derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Tregs), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils that can mediate a cytotoxic response.

[0250] In some examples, the useful cells expressing the multispecific antibodies of the present disclosure can include producer T cells. Non-limiting examples of producer T cells include those described in Tsai & Davila Oncoimmunology. (2016) 5(5): e1122158, the disclosure of which is incorporated herein by reference in its entirety. Producer T cells engineered to contain a nucleic acid sequence encoding the multispecific antibody of the present disclosure can, in some examples, be used to deliver the antibody to a subject in need thereof.

[0251] The cells of the present disclosure also include cells genetically modified to change and / or modify the expression of one or more of MDR1 and CD47 in the cells. Such modified cells are useful for various purposes, including assaying the binding of multispecific antibodies produced according to the descriptions and methods provided herein, but not limited thereto. Optionally, MDR1 can be knocked out or knocked down in the target cell line. Optionally, CD47 can be knocked out or knocked down in the target cell line. In some examples, MDR1 can be constitutively or inducibly overexpressed in the target cell line. In some examples, CD47 can be constitutively or inducibly overexpressed in the target cell line. In some examples, both MDR1 and CD47 can be knocked down, knocked out, or constitutively or inducibly overexpressed in the target cell line. Any convenient and appropriate method for knockdown, knockout, and / or overexpression can be used. The introduced nucleic acid can be stably incorporated or can be transiently present.

[0252] In some embodiments, the cells of the present disclosure include a genetically engineered human cell line that expresses CD47 and includes an exogenous nucleic acid encoding MDR1 for overexpression of MDR1. In such cells, CD47 expression can be endogenous or exogenous (i.e., introduced), and MDR1 expression can be stable or transient. In some examples, the cell line of the present disclosure that expresses CD47 can be configured to produce a genetically engineered human cell that expresses CD47 and stably overexpresses MDR1.

[0253] The cells and cell lines of the present disclosure can be cultured, for example, via the use of the culture methods described herein. Optionally, a cell line can be generated by culturing cells that have been genetically modified by introducing a nucleic acid. Useful cell lines can include, but are not limited to, genetically engineered cell lines, such as, for example, a human cell line that expresses CD47 and stably overexpresses MDR1.

[0254] The cells and cell lines of the present disclosure can be used as, for example, test samples, controls, etc. in various methods of the present disclosure. For example, in some examples, cells in which MDR1 and / or CD47 are knocked out and / or knocked down can be used as reference cells, for example, against which the binding of the multispecific antibodies of the present disclosure can be compared. Other useful reference cells include, for example, non-cancerous cells, as well as normal cells and cells that express normal levels of various proteins (including normal levels of MDR1 and / or CD47), but are not limited thereto.

[0255] [Method] As summarized above, the methods of the present disclosure include methods of contacting cells with the antibodies of the present disclosure, methods of treating a subject according to methods that include administering the antibodies of the present disclosure to the subject, methods of making the elements described in the present application (including, for example, multispecific antibodies, compositions and formulations, nucleic acids, expression vectors, cells, etc.).

[0256] As summarized above, the methods of the present disclosure include contacting cancer cells with the multispecific antibodies of the present disclosure, for example, to promote and / or enhance the killing of cancer cells. In some examples, the killing of cancer cells is mediated by an immune response or immune cells that act on the cancer cells as a result of opsonization of the cancer cells by bispecific targeting when the two targets are co-expressed on the cancer cells. In some examples, the killing of cancer cells is mediated by an immune response or immune cells that act on the cancer cells as a result of masking or antagonizing a CD47 epitope present on the surface of the cancer cells by, for example, a multispecific antibody. In some examples, the killing of cancer cells is mediated by inhibition of cancer cell efflux as a result of, for example, MDR1 antagonism on the cancer cells by a multispecific antibody. In some cases, the cells contacted with the multispecific antibody can be multidrug-resistant cancer cells. The method of contacting cancer cells with the multispecific antibody of the present disclosure may or may not include contacting the cancer cells with additional therapies or active agents, including, for example, chemotherapy, immunotherapy, radiation therapy, etc.

[0257] Contacting cancer cells with the multispecific antibodies of the present disclosure generally enhances the killing of cancer cells (e.g., as compared to the level of cancer cell killing in the absence of the multispecific antibody). In some examples, when an additional active agent is used, an enhancement of cancer cell killing can be seen as compared to the level of killing observed using the additional active agent alone. The amount of enhancement of cancer cell killing due to the multispecific antibody can vary and can range from at least a 5% increase to at least a 90% or greater increase in cancer cell killing, for example, including but not limited to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, etc. Such an increase can be compared to single contact with one or more additional active agents.

[0258] The enhanced killing of cancer cells can be evaluated by various means including, but not limited to, observational studies, in vitro cell-based cytotoxicity assays, flow cytometry, cell viability markers (e.g., those using one or more cell viability stains), etc.

[0259] [Treatment method] The present disclosure provides a method for treating cancer, the method generally comprising administering to an individual in need thereof (e.g., a person having cancer) an effective amount of the subject multispecific antibody, alone (e.g., as a monotherapy), or in combination with one or more additional therapeutic agents (e.g., as a combination therapy). Administration of the multispecific antibodies of the present disclosure can be effected by any convenient and appropriate route of delivery.

[0260] Aspects of the present disclosure include bispecific antibody molecules according to the preceding sections of this specification for use in a method of treating cancer in a subject, the method including administering the antibody to the subject. The method includes administering the antibody in combination with at least one additional active agent, where the at least one additional active agent includes a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. In certain aspects, the at least one additional active agent is a chemotherapeutic agent, and optionally the chemotherapeutic agent is paclitaxel, a vinca alkaloid, or an anthracycline. Some chemotherapeutic agents that are substrates of the MDR1 pump include paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide, and nilotinib.

[0261] Also disclosed herein is a chemotherapeutic agent for use in a method of treating cancer in a subject, the method including administering the chemotherapeutic agent to the subject in combination with an antibody described herein, and optionally the chemotherapeutic agent is paclitaxel, a vinca alkaloid, or an anthracycline.

[0262] Thus, administering can include, for example, delivery of the antibody by injection, delivery of the antibody by infusion, delivery of a nucleic acid or expression vector encoding the multispecific antibody, delivery of the antibody by administering to the subject cells that express and secrete the multispecific antibody, but is not limited thereto. Administration of an agent, a nucleic acid encoding the agent, a cell expressing the agent, etc. can include contacting the agent, contacting the nucleic acid, contacting the cell, etc.

[0263] In some embodiments, the effective amount of the target bispecific antibody, when administered alone (e.g., as monotherapy) or in combination with one or more additional therapeutic agents (e.g., as combination therapy) in one or more doses, reduces the severity of the adverse symptoms of cancer by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, as compared to the severity of the adverse symptoms in the absence of treatment with the antibody.

[0264] In one aspect, the effective amount of the target bispecific antibody, when administered alone (e.g., as monotherapy) or in combination with one or more additional therapeutic agents (e.g., as combination therapy) in one or more doses, is an amount effective to improve cancer (e.g., slow cancer growth, stop cancer growth, reverse cancer growth, kill cancer cells (including tumor cells, etc.)) in the individual being treated. For example, the effective amount of the antibody of interest is 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, effective to reduce the cancer growth rate or reduce the size of the cancer in the individual, as compared to the absence of treatment with the bispecific antibody.

[0265] In some instances, the subject can be treated systemically, with or without one or more additional reagents, including using the target bispecific antibody. As used herein, "systemic treatment" means a treatment that does not target only a specific tumor (e.g., a primary tumor or a defined secondary tumor, etc.) or a specific cancer-containing tissue (e.g., the liver in the case of liver cancer, the blood in the case of blood cancer, etc.). Systemic treatment is generally directed to the entire body of the subject and can include, but is not limited to, whole body radiotherapy, whole body chemotherapy, whole body immunotherapy, combinations thereof, and the like.

[0266] In some examples, the subject can be treated locally, including with or without one or more additional reagents, using a subject - specific antibody. As used herein, "local treatment" means a treatment specifically directed to the location of a tumor (e.g., a primary tumor or a defined secondary tumor, etc.), or a treatment specifically directed to a cancer - containing tissue (e.g., the liver in the case of liver cancer, blood in the case of blood cancer, etc.). In some examples, the local treatment can also be administered in a manner that affects the environment surrounding the tumor (e.g., the tissue surrounding the tumor, e.g., the tissue immediately adjacent to the tumor). Local treatment generally does not affect or target tissues distant from the cancer site that includes the site of the tumor (e.g., the primary tumor). Useful local treatments that can be administered in addition to or in combination with the subject - specific antibody include, but are not limited to, for example, surgery, local radiotherapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, etc.

[0267] In one aspect, the subject treatment method includes administering a subject - specific antibody and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapy reagents, immunotherapy reagents, other antibodies or bispecific antibody agents, etc. The additional treatments that can be administered to the subject before, during, or after administration of the bispecific antibody of the present disclosure vary depending on a number of factors including, for example, the type of cancer, the subject's medical history, general health status, and / or co - morbidities. Useful cancer treatments include, but are not limited to, for example, radiotherapy, chemotherapy, immunotherapy, etc.

[0268] Radiotherapy includes, but is not limited to, X - rays or gamma rays delivered from an external source applied like a beam or by implantation of small radioactive sources.

[0269] Antibodies suitable for use in cancer treatment include naked antibodies, such as trastuzumab (Herceptin), bevacizumab (Avastin (trademark)), cetuximab (Erbitux (trademark)), panitumumab (Vectibix (trademark)), ipilimumab (Yervoy (trademark)), rituximab (Rituxan), alemtuzumab (Lemtrada (trademark)), ofatumumab (Arzerra (trademark)), oregovomab (OvaRex (trademark)), pembrolizumab (MK-3475), pertuzumab (Perjeta (trademark)), ranibizumab (Lucentis (trademark)), and conjugated antibodies, such as gemtuzumab ozogamicin (Mylotarg (trademark)), brentuximab vedotin (Adcetris (trademark)), 90Y-labeled ibritumomab tiuxetan ((Zevalin (trademark)), 131I-labeled tositumomab (Bexxar (trademark)), etc., but are not limited thereto. Antibodies suitable for use in cancer treatment also include, but are not limited to, antibodies produced against tumor-associated antigens. Such antigens include CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Le y, VEGF, VEGFR, integrin alpha-V-beta-3, integrin alpha-5-beta-1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, etc., but are not limited thereto.

[0270] Conventional cancer therapies include targeted therapies for cancer, such as but not limited to the following: Ado-trastuzumab emtansine (Kadcyla) targeting HER2 (ERBB2 / neu) (approved for use in breast cancer); afatinib (Gilotrif) targeting EGFR (HER1 / ERBB1) and HER2 (ERBB2 / neu) (approved for use in non-small cell lung cancer); Aldesleukin (Proleukin) for targeting (approved for use in renal cell carcinoma and melanoma); alectinib (Alecensa) targeting ALK (approved for use in non-small cell lung cancer); alemtuzumab (Campath) targeting CD52 (approved for use in B-cell chronic lymphocytic leukemia); atezolizumab (Tecentriq) targeting PD-L1 (approved for use in urothelial cancer and non-small cell lung cancer); avelumab (Bavencio) targeting PD-L1 (approved for use in Merkel cell carcinoma); axitinib (Inlyta) targeting KIT, PDGFRβ, VEGFR1 / 2 / 3 (approved for use in renal cell carcinoma); belimumab (Benlysta) targeting BAFF (approved for use in lupus erythematosus); belinostat (Beleodaq) targeting HDAC (approved for use in peripheral T-cell lymphoma); bevacizumab (Avastin) targeting VEGF ligand (approved for use in cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma, non-small cell lung cancer; ovarian cancer, peritoneal cancer, renal cell carcinoma); blinatumomab (Blincyto) targeting CD19 / CD3 (approved for use in acute lymphoblastic leukemia (precursor B-cell)); bortezomib (Velcade) targeting proteasome (approved for use in multiple myeloma and mantle cell lymphoma); bosutinib (Bosulif) targeting ABL (approved for use in chronic myeloid leukemia); brentuximab vedotin (Adcetris) targeting CD30 (approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma); brigatinib (Alunbrig) targeting ALK (approved for use in non-small cell lung cancer (ALK+));Cabozantinib (Cabometyx, Cometriq), which targets FLT3, KIT, MET, RET, VEGFR2 (approved for use in medullary thyroid cancer and renal cell carcinoma); Carfilzomib (Kyprolis), which targets the proteasome (approved for use in multiple myeloma); Crizotinib (Xalkori), which targets ALK, MET, ROS1 (approved for use in non-small cell lung cancer); Cetuximab (Erbitux), which targets EGFR (HER1 / ERBB1) (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); Dabrafenib (Tafinlar), which targets BRAF (approved for use in melanoma and non-small cell lung cancer); Daratumumab (Darzalex), which targets CD38 (approved for use in multiple myeloma); Dasatinib (Sprycel), which targets ABL (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); Denosumab (Xgeva), which targets RANKL (approved for use in giant cell tumor of bone); Dinutuximab (Unituxin), which targets B4GALNT1 (GD2) (approved for use in pediatric neuroblastoma); Durvalumab (Imfinzi), which targets PD-L1 (approved for use in urothelial carcinoma); Elotuzumab (Empliciti), which targets SLAMF7 (CS1 / CD319 / CRACC) (approved for use in multiple myeloma); Enasidenib (Idhifa), which targets IDH2 (approved for use in acute myeloid leukemia); Erlotinib (Tarceva), which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer and pancreatic cancer); Everolimus (Afinitor), which targets mTOR (approved for use in neuroendocrine tumors of pancreatic, gastrointestinal, or lung origin, renal cell carcinoma, unresectable ependymoma, and breast cancer); Gefitinib (Iressa), which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer);Ibritumomab tiuxetan (Zevalin), targeting CD20 (approved for use in non-Hodgkin lymphoma); Ibrutinib (Imbruvica), targeting BTK (approved for use in mantle cell lymphoma, chronic lymphocytic leukemia, Waldenström macroglobulinemia); Idelalisib (Zydelig), targeting PI3Kδ (approved for use in chronic lymphocytic leukemia, follicular B-cell non-Hodgkin lymphoma, small lymphocytic lymphoma); Imatinib (Gleevec), targeting KIT, PDGFR, ABL (approved for use in gastrointestinal stromal tumor (KIT+), dermatofibrosarcoma protuberans, multiple hematological tumors); Ipilimumab (Yervoy), targeting CTLA-4 (approved for use in melanoma); Ixazomib (Ninlaro), targeting proteasome (approved for use in multiple myeloma); Lapatinib (Tykerb), targeting HER2 (ERBB2 / neu), EGFR (HER1 / ERBB1) (approved for use in breast cancer (HER2+)); Lenvatinib (Lenvima), targeting VEGFR2 (approved for use in renal cell carcinoma, thyroid cancer); Midostaurin (Rydapt), targeting FLT3 (approved for use in acute myeloid leukemia (FLT3+)); Necitumumab (Portrazza), targeting EGFR (HER1 / ERBB1) (approved for use in squamous non-small cell lung cancer); Neratinib (Nerlynx), targeting HER2 (ERBB2 / neu) (approved for use in breast cancer); Nilotinib (Tasigna), targeting ABL (approved for use in chronic myeloid leukemia); Niraparib (Zejula), targeting PARP (approved for use in ovarian cancer, fallopian tube cancer, peritoneal cancer); Nivolumab (Opdivo), targeting PD-1 (approved for use in colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, melanoma, non-small cell lung cancer, renal cell carcinoma, urothelial cancer); Obinutuzumab (Gazyva), targeting CD20 (approved for use in chronic lymphocytic leukemia, follicular lymphoma); Ofatumumab (Arzerra, HuMax-CD20), targeting CD20 (approved for use in chronic lymphocytic leukemia);Olaparib (Lynparza), which targets PARP (approved for use in ovarian cancer); Olalizumab (Lartruvo), which targets PDGFRα (approved for use in soft tissue sarcoma); Osimertinib (Tagrisso), which targets EGFR (approved for use in non-small cell lung cancer); Palbociclib (Ibrance), which targets CDK4 and CDK6 (approved for use in breast cancer); Panitumumab (Vectibix), which targets EGFR (HER1 / ERBB1) (approved for use in colorectal cancer); Panobinostat (Farydak), which targets HDAC (approved for use in multiple myeloma); Pazopanib (Votrient), which targets VEGFR, PDGFR, and KIT (approved for use in renal cell carcinoma); Pembrolizumab (Keytruda), which targets PD-1 (approved for use in classical Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+), head and neck squamous cell carcinoma, solid tumors (MSI-H)); Pertuzumab (Perjeta), which targets HER2 (ERBB2 / neu) (approved for use in breast cancer (HER2+)); Ponatinib (Iclusig), which targets ABL, FGFR1-3, FLT3, and VEGFR2 (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); Ramucirumab (Cyramza), which targets VEGFR2 (approved for use in colorectal cancer, gastric cancer, or gastroesophageal junction (GEJ) adenocarcinoma, and non-small cell lung cancer); Regorafenib (Stivarga), which targets KIT, PDGFRβ, RAF, RET, and VEGFR1 / 2 / 3 (approved for use in colorectal cancer, gastrointestinal stromal tumor, and hepatocellular carcinoma); Ribociclib (Kisqali), which targets CDK4 and CDK6 (approved for use in breast cancer (HR+, HER2-)); Rituximab (Rituxan, MabThera), which targets CD20 (approved for use in non-Hodgkin lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis); Rituximab / Hyaluronidase, Human (Rituxan Hycela), which targets CD20 (approved for use in chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma);Romidepsin (Istodax), which targets HDAC (approved for use in cutaneous T-cell lymphoma and peripheral T-cell lymphoma); Rucaparib (Rubraca), which targets PARP (approved for use in ovarian cancer); Ruxolitinib (Jakafi), which targets JAK1 / 2 (approved for use in myelofibrosis); Siltuximab (Sylvant), which targets IL-6 (approved for use in multicentric Castleman disease); Targeted Sipuleucel-T (Provenge) (approved for use in prostate cancer); Sonidegib (Odomzo), which targets smoothened (approved for use in basal cell carcinoma); Sorafenib (Nexavar), which targets VEGFR, PDGFR, KIT, RAF (approved for use in hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer); Temsirolimus (Torisel), which targets mTOR (approved for use in renal cell carcinoma); Tositumomab (Bexxar), which targets CD20 (approved for use in non-Hodgkin lymphoma); Trametinib (Mekinist), which targets MEK (approved for use in melanoma and non-small cell lung cancer); Trastuzumab (Herceptin), which targets HER2 (ERBB2 / neu) (approved for use in breast cancer (HER2+) and gastric cancer (HER2+)); Vandetanib (Caprelsa), which targets EGFR (HER1 / ERBB1), RET, VEGFR2 (approved for use in medullary thyroid cancer); Vemurafenib (Zelboraf), which targets BRAF (approved for use in melanoma); Venetoclax (Venclexta), which targets BCL2 (approved for use in chronic lymphocytic leukemia); Vismodegib (Erivedge), which targets PTCH and smoothened (approved for use in basal cell carcinoma); Vorinostat (Zolinza), which targets HDAC (approved for use in cutaneous T-cell lymphoma); Ziv-aflibercept (Zaltrap), which targets PIGF, VEGFA / B (approved for use in colorectal cancer); etc.;

[0271] 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) serine / threonine kinase activity inhibitors; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon-α; (7) interferon-γ; (8) colony-stimulating factors; (9) angiogenesis inhibitors; and (10) antagonists of tumor necrosis factor.

[0272] Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous) compounds that reduce the growth of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0273] Agents that act to reduce cell growth are known in the art and are widely used. Such agents include, but are not limited to, alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including mechlorethamine, cyclophosphamide (Cytoxan (trademark)), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chloromethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, dacarbazine, and temozolomide.

[0274] Antimetabolites include, but are not limited to, folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, such as cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0275] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, epipodophyllotoxins) include Ara-C, paclitaxel (Taxol (registered trademark)), docetaxel (Taxotere (registered trademark)), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids such as vincristine, vinblastine, vinorelbine, vindesine; podophyllotoxins such as etoposide, teniposide; antibiotics such as anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives; phenoxazone bicyclic peptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrrolindiones such as mitomycin; macrolide immunosuppressants such as cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin; etc., but are not limited thereto.

[0276] Other antiproliferative cytotoxic agents are navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, cyclophosphamide, ifosfamide, and droloxifene.

[0277] Microtubule-acting agents having antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), halicondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), lysocine (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, including but not limited to natural and synthetic epothilones such as epothilone A, epothilone B, discodermolide; estramustine, nocodazole, etc.

[0278] Suitable hormonal regulators and steroids (including synthetic analogs) include corticosteroids such as prednisone, dexamethasone; estrogens and progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, etc.; and adrenocortical suppressants such as aminoglutethimide; 17α-ethinyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, drostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex, etc., but are not limited to these. Since estrogen stimulates growth and differentiation, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit the proliferation of T cells.

[0279] Other chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, etc.; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epipodophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, etc. Other growth inhibitors include immunosuppressants such as mycophenolic acid, thalidomide, deoxyspergualin, azathioprine, leflunomide, mizoribine, azaspiracid (SKF 105685), etc.; Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc.

[0280] "Taxane" includes, in addition to paclitaxel, active taxane derivatives or prodrugs. "Paclitaxel" (for example, docetaxel, Taxol® trademark, Taxotere® trademark (a formulation of docetaxel), analogs such as 10-desacetyl analogs of paclitaxel and 3'N-desbenzoyl-3'N-t-butoxycarbonyl analogs of paclitaxel, formulations and derivatives, should be understood herein to be included) can be readily prepared using techniques known to those skilled in the art (see also 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; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or can be obtained from various commercial sources such as, for example, Sigma Chemical Co. of St. Louis, Missouri (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis).

[0281] Paclitaxel is to be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogs and derivatives (such as the above-mentioned Taxotere (trademark) docetaxel) and paclitaxel conjugates (such as paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, or paclitaxel-albumin).

[0282] Also, the term "taxane" includes various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application WO 99 / 18113; the piperazino and other derivatives described in WO 99 / 14209; the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO 98 / 28288; the sulfenamide derivatives described in U.S. Patent No. 5,821,263; and the taxol derivatives described in U.S. Patent No. 5,415,869. Further included, but not limited to, are the prodrugs of paclitaxel described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0283] Useful immunotherapies include the following: anti-PD-1 / PD-L1 immunotherapy and / or other immunotherapy targets that can be targeted in the treatment method, such as immune checkpoint markers such as CTLA-4, LAG-3, and TIM-3. Anti-PD-1 / PD-L1 immunotherapy includes, for example, but is not limited to, therapies that include administering to a subject an effective amount of one or more anti-PD-1 / PD-L1 therapeutic antagonists, such antagonists including, for example, Opdivo® (nivolumab), Keytruda® (pembrolizumab), Tecentriq® (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242, etc., but not limited thereto.

[0284] CTLA-4, also known as CD152, binds to CD80 and CD86. Antibodies against CTLA-4 have been approved as therapeutic agents for some types of cancer. Due to its co-inhibitory effect with other immunotherapies, CTLA-4 is a good candidate for use in combination with other immunotherapies to treat certain cancers. TIM-3 can also be targeted for immunotherapy against some cancer types.

[0285] LAG-3 is in clinical trials for cancer treatment. Anti-LAG-3 immunotherapy includes those using antagonist LAG-3 antibodies that can (by downregulating LAG-3 and inhibiting the signal to make pre-activated LAG-3+ cells) activate T effector cells while inhibiting the induced (i.e., antigen-specific) Treg inhibitory activity. Useful LAG-3 antagonist antibodies include relatlimab (BMS-986016; developed by Bristol-Myers Squibb), IMP701 (developed by Immutep), TSR-033 (anti-LAG-3 mAb; developed by TESARO, Inc.), etc.

[0286] Immunotherapies also include T cell-based immunotherapies such as adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T cell therapy. For example, a population of CAR T cells engineered to target an antigen expressed by the cancer of a subject can be administered to the subject. T cell-based therapies can, in some instances, involve obtaining a cell sample, such as a blood sample or a tumor biopsy, from the subject and culturing immune cells ex vivo from the sample, with or without genetic modification of the cultured immune cells. As an example, immune cells can be obtained from a subject, cultured ex vivo, and modified with a CAR specific for an antigen expressed by the cancer to produce a population of CAR T cells. The CAR T cells can then be reintroduced into the subject to target the cancer. T cell-based immunotherapies can be configured in a variety of ways depending on the particular cancer being treated, such as targeting various antigens, collecting / culturing various cell types, etc. Further, T cell-based immunotherapies can be administered systemically (e.g., by intravenous injection) or locally (e.g., by infusion (e.g., intraperitoneal infusion, intrathoracic catheter infusion, etc.), direct injection, etc.).

[0287] In some instances, the treatment methods described herein can include administering to a subject one or more inhibitors of multidrug resistance transporters, which can include, but are not limited to, multidrug resistance transporters other than MDR1. Useful inhibitors of multidrug resistance transporters include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and small molecule inhibitors. Inhibitors of multidrug resistance transporters include ABC transporter inhibitors. A summary of such MDR modulators or reversers is provided in Choi (2005), Cancer Cell Int, 5:30, the disclosure of which is incorporated herein by reference in its entirety.

[0288] Individuals suitable for treatment using the methods of the present disclosure include individuals with cancer; individuals diagnosed with cancer; individuals undergoing cancer treatment by chemotherapy, radiotherapy, antibody therapy, surgery, etc.; individuals who have undergone cancer treatment (e.g., by one or more of chemotherapy, radiotherapy, antibody therapy, surgery, etc.) but did not respond to the treatment; individuals who have undergone cancer treatment (e.g., by one or more of chemotherapy, radiotherapy, antibody therapy, surgery, etc.) and initially responded to the treatment but then relapsed, i.e., the cancer has recurred.

[0289] The methods of the present disclosure can be used, for example, to target and treat various cancers, including, but not limited to, primary cancer, secondary cancer, regrowth cancer, recurrent cancer, refractory cancer, etc. For example, in some instances, the methods of the present disclosure can be used as an initial treatment for primary cancer identified in a subject. In some instances, the methods of the present disclosure can be used as a non-primary (e.g., secondary or later) treatment, for example, in a subject with cancer that did not respond to a previous treatment, in a subject with cancer that has regrown after a previous treatment, or in a subject having a mixed response to a previous treatment (e.g., a positive response to at least one tumor in the subject and a negative or neutral response to at least one second tumor in the subject).

[0290] In some instances, the methods of the present disclosure can be used to treat subjects having drug-resistant cancers, such as multi-drug resistant cancers. Multi-drug resistance (MDR) is a mechanism by which many cancers develop resistance to chemotherapeutic drugs, minimizing cell death and expanding drug-resistant tumors. MDR cancers can involve one or more resistance mechanisms, including, but not limited to, for example, increased expression of efflux pumps, decreased drug uptake, inhibition of cell death or apoptosis, regulation of drug metabolism, etc. In some instances, the methods of the present disclosure can prevent, reverse, or avoid MDR.

[0291] In some examples, the methods of the disclosure can include treating a subject having cancer that is resistant to a first agent, with an effective amount of the multispecific antibody described herein, in combination with a second agent that is different from the first agent. For example, in some examples, the subject's cancer can be resistant to a first chemotherapy, and the subject can be treated by administering an effective amount of the multispecific antibody described herein, in combination with a second chemotherapeutic agent that is different from the first. For example, depending on the type of cancer being treated, the likelihood of developing resistance, etc., various combinations of first and second chemotherapeutic agents can be used.

[0292] A number of cancers are known to develop drug resistance. For this and other reasons, the methods of the disclosure can find use in treating a variety of cancers including, but not limited to, for example, Acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer (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 tumor, craniopharyngioma, ependymoma, etc.), breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumor, Burkitt lymphoma, carcinoid tumor (e.g., pediatric, gastrointestinal tract, etc.), carcinoma of unknown primary, heart (cardiac) tumor, central nervous system (e.g., atypical teratoid / rhabdoid tumor, embryonal tumor, germ cell tumor, lymphoma, etc.), cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, duct (e.g., bile duct, extrahepatic, etc.), ductal carcinoma in situ (DCIS), fetal tumor, endometrial cancer, ependymoma, esophageal cancer, neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), fibrous histiocytoma of bone (e.g., malignant, osteosarcoma, etc.), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) carcinoma, histiocytosis (e.g., Langerhans cell, etc.), Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (e.g., pancreatic neuroendocrine tumor, etc.), Kaposi's sarcoma, kidney cancer (e.g., renal cell, Wilms tumor, pediatric renal tumor, etc.), Langerhans cell histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell, etc.), lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), lymphoma (e.g., AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system (CNS), etc.), macroglobulinemia(e.g., Waldenström, etc.), male breast cancer, malignant fibrous histiocytoma and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary origin, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplasia / myeloproliferative neoplasm, myeloid leukemia (e.g., chronic (CML), etc.), myeloblastic leukemia (e.g., acute (AML), etc.), myeloproliferative neoplasm (e.g., chronic, etc.), nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cavity cancer, oral cancer (e.g., lip, etc.), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer (e.g., epithelial, germ cell tumor, low-grade tumor, etc.), pancreatic cancer, pancreatic neuroendocrine tumor (islet cell tumor), papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis / ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue, uterine, etc.), Sézary syndrome, skin cancer (e.g., childhood, melanoma, Merkel cell carcinoma, non-melanoma, etc.), small cell lung cancer, small intestine cancer, soft tissue, squamous cell carcinoma, squamous cell carcinoma of the neck (e.g., of unknown primary origin, metastatic, etc.), stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymocyte tumor, thyroid cancer, transitional cell carcinoma of the renal pelvis / ureter, ureter / pelvis cancer, urethral cancer, uterine cancer (e.g., endometrial, etc.), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor, etc.

[0293] The treatment methods described herein can, in some instances, be carried out in subjects who have previously received one or more conventional treatments. For example, in the case of oncology, the methods described herein can, in some instances, be carried out after conventional cancer treatments (e.g., including, but not limited to, conventional chemotherapy, conventional radiotherapy, conventional immunotherapy, surgery, etc.). In some instances, the methods described herein can be used when a subject has not responded to conventional treatment or is refractory. In some examples, the methods described herein can be used when a subject has responded to conventional treatment.

[0294] In some examples, the methods of the disclosure can be used to target, treat, or eliminate minimal residual disease (MRD) remaining after a prior cancer treatment in a subject. Targeting, treating, and / or clearance of MRD can be pursued using the methods herein, regardless of whether the MRD is refractory or determined to be refractory to the prior treatment. In some examples, the methods of the disclosure can be used to target, treat, and / or eliminate MRD in a subject after a determination that the MRD is refractory to a prior treatment or one or more available treatment options other than those using the multispecific antibodies described herein.

[0295] In some instances, the methods of the invention can be used prophylactically for monitoring. For example, a subject in need thereof can receive administration of a treatment comprising one or more of the multispecific antibodies described herein when the subject does not have a detectable disease but is at risk of developing recurrent cancer (including, e.g., drug-resistant cancer). In some examples, a prophylactic approach can be used when a subject has a particularly high risk of developing a primary cancer that is predicted to be drug-resistant or become drug-resistant. In some cases, a prophylactic approach can be employed when a subject has previously received treatment for cancer and is at risk of recurrence or development of drug resistance.

[0296] In some examples, the methods of the disclosure may include analyzing cancer for the expression of one or more markers or therapeutic targets. For example, in some instances, the method may include analyzing a sample of cancer from a subject to determine whether the cancer expresses MDR1 above a predetermined threshold, TAA (e.g., CD47, PD-L1, or EGFR) above a predetermined threshold, or both.

[0297] In some examples, whether a subject is to be treated with the multispecific antibodies of the disclosure may depend on the results of the TAA and / or MDR1 tests. For example, in some instances, if the cancer expresses TAA above a predetermined threshold, the subject may be treated with the multispecific antibodies of the disclosure, and if the cancer expresses TAA below a predetermined threshold, the subject may not be treated with the multispecific antibodies; for example, the subject may be treated with a conventional treatment for the relevant cancer without the multispecific antibodies. In some cases, if the cancer expresses MDR1 above a predetermined threshold, the subject may be treated with the multispecific antibodies of the disclosure, and if the cancer expresses MDR1 below a predetermined threshold, the subject may not be treated with the multispecific antibodies; for example, the subject may be treated with a conventional treatment for the relevant cancer without the multispecific antibodies. In some cases, if the cancer expresses both TAA and MDR1 above a predetermined threshold, the subject may be treated with the multispecific antibodies of the disclosure, and if the cancer expresses both TAA and MDR1 below a predetermined threshold, the subject may not be treated with the multispecific antibodies; for example, the subject may be treated with a conventional treatment for the relevant cancer without the multispecific antibodies.

[0298] To analyze the levels of MDR1 and / or TAA, any convenient assay can be used, including but not limited to, for example, flow cytometry, nucleic acid-based assays (such as amplification, sequencing, etc.), cytometric analysis, immunohistochemistry, etc. Any convenient biological sample can be used, including but not limited to, for example, cancer biopsy samples. A useful predetermined threshold for evaluating the expression of one or more markers and / or targets can be determined by any convenient and appropriate method, including comparison of the measured expression level with a corresponding control. For example, in some instances, a useful predetermined threshold for the levels of MDR1 and / or TAA assayed in a sample can correspond to the levels of MDR1 and / or TAA measured in reference cells such as healthy / normal cells. The TAA can be CD47, PD-L1, or EGFR.

[0299] [Method of manufacture] As summarized above, the methods of the disclosure also include methods for making and / or identifying the multispecific antibodies described herein. The subject antibodies can be made by known methods, such as conventional synthetic methods for protein synthesis; recombinant DNA methods; etc.

[0300] When the antibody of interest is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, the synthesis can proceed via a liquid or solid phase. Solid-phase polypeptide synthesis (SPPS) is an example of a suitable method for the chemical synthesis of the antibody of interest, where 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, such as Fmoc and Boc, are available for synthesizing the antibody of interest. Techniques for solid-phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. Briefly, small insoluble porous beads are treated in a functional unit where the peptide chain is constructed. After repeated cycles of coupling / deprotection, the free N-terminal amine bound to the solid phase is coupled to a single N-protected amino acid unit. This unit is then deprotected, exposing a new N-terminal amine to which additional amino acids can bind. The peptide remains immobilized on the solid phase and is cleaved after undergoing a filtration process.

[0301] Standard recombinant methods can be used to produce the antibody of interest. For example, nucleic acids encoding the light and heavy chain variable regions (optionally linked to constant regions) are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in an expression vector (s) that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, enhancer elements, and transcription termination sequences. The expression control sequences can be eukaryotic promoter systems in vectors that can transform or transfect eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of its nucleotide sequence as well as collection and purification of the antibody.

[0302] Due to the degeneracy of the code, various nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be generated by de novo solid phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of a previously prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer and encodes the selected modification in the target polypeptide DNA.

[0303] Suitable expression vectors are typically capable of replicating episomally in the host organism or as an integrated part of the host chromosomal DNA. Generally, expression vectors contain a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) that enables the detection of cells transformed with the desired DNA sequence.

[0304] Escherichia coli is an example of a prokaryotic host cell that can be used to clone polynucleotides encoding the antibody of interest. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be constructed, which typically contain expression control sequences compatible with the host cell (e.g., origin of replication). Furthermore, there are any number of various well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system derived from phage lambda. The promoter typically controls expression, optionally with an operator sequence, and has a ribosome binding site sequence, etc. to initiate and complete transcription and translation.

[0305] 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 have expression control sequences (e.g., promoters), origins of replication, termination sequences, etc. as required. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, cytochrome C, and enzymes involved in maltose and galactose utilization.

[0306] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce the polypeptides of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and immortalized B cells or hybridomas. Expression vectors for these cells can include expression control sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0307] Once synthesized (either chemically or by recombinant techniques), whole antibodies, their dimers, individual light and heavy chains, or other forms of the subject antibodies (e.g., scFv, etc.) can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (generally see Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). The subject antibodies can be substantially pure, e.g., at least about 80% - 85% pure, at least about 85% - 90% pure, at least about 90% - 95% pure, or 98% - 99%, or more pure, e.g., free of contaminants such as cell debris, macromolecules other than the subject antibody.

[0308] In some embodiments, methods of making the multispecific antibodies of the present disclosure can include making candidate antibodies and screening for activity. Such methods can generate multispecific antibodies that specifically bind to cells that express both MDR1 and CD47 through the use of a series of steps. The steps of such methods include producing a multispecific antibody, or a plurality of antibodies each of which is expected to include or include an MDR1 binding domain and a CD47 binding domain; contacting a first test cell expressing MDR1 and CD47 with the multispecific antibody or the plurality of antibodies; contacting a second cell expressing either MDR1 or CD47 with the multispecific antibody or the plurality of antibodies; determining a binding specificity ratio by comparing the binding of the multispecific antibody or the plurality of antibodies to the first cell with the binding of the multispecific antibody to the second cell; and identifying one or more of the multispecific antibody or the plurality of antibodies as being specific for cells that express both MDR1 and CD47 if the ratio exceeds a predetermined threshold. When such a threshold for comparative binding is used, the threshold can vary and can be in the range of 1.5:1 or greater, for example, including, but not limited to, 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.

[0309] In such methods, a variety of cells can be used, including but not limited to, for example, the cells described herein. In some examples, antibody binding can be performed against both cells that express only MDR1 and cells that express only CD47. For example, in some examples, the method can further include contacting the multispecific antibody with a third cell that expresses CD47 but not MDR1 in the case where the second cell expresses MDR1 but not CD47, in connection with the steps described above.

[0310] In some examples, such methods may use one or more controls, including but not limited to, for example, control cells, control reagents, etc. Useful control cells include those in which the expression of one or more related genes or proteins is known or the absence of such expression is known. Useful control reagents can include, but are not limited to, control antibodies such as monoclonal antibodies against known targets. For example, in some examples, such methods of the present disclosure may further include contacting a first cell, a second cell, and / or a third cell with a control antibody selected from a monoclonal anti-MDR1 antibody and a monoclonal anti-CD47 antibody. Depending on the particular method used, various other or additional controls may be used as needed.

[0311] [Kit] Aspects of the present disclosure also include kits. The kits can include, for example, any combination of multispecific antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. described herein. The subject kits can include one or more of the multispecific antibody, the nucleic acid encoding it, or the cells containing the multispecific nucleic acid. The kits can be configured for various purposes, including, for example, therapeutic kits (e.g., where the kit can include a multispecific antibody and one or more additional active agents such as, for example, chemotherapeutic agents), kits for producing antibodies, kits for screening antibodies, etc.

[0312] The optional components of the kits are diverse and can include, for example: buffers; protease inhibitors; etc. For example, if the subject kit contains a subject nucleic acid, the nucleic acid may have restriction sites, multiple cloning sites, primer sites, etc. The various components of the kit may be present in separate containers or, if desired, certain compatible components may be pre-combined in a single container.

[0313] In addition to the above components, the kit can include instructions for using the components of the kit to carry out the method. Instructions for carrying out the method of interest are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. As such, the instructions may be present as a package insert, on the label of the kit or a container of its components (i.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file on a suitable computer-readable storage medium, such as a compact disc read-only memory (CD-ROM), digital versatile disc (DVD), floppy disk, etc. In still other embodiments, the actual instructions are not present in the kit, but means are provided for obtaining the instructions from a remote source, e.g., via the Internet. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0314] [Examples of non-limiting aspects of the present disclosure] The aspects (including embodiments) of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, specific non-limiting aspects of the present disclosure are provided below. As will be apparent to those skilled in the art upon reading the present disclosure, each of the individually numbered aspects can be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of multiple aspects and is not limited to the combinations of aspects explicitly provided below. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the present invention.

[0315] 1. A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and tumor-associated antigen (TAA), comprising two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain, wherein each of said VL chains comprises an antigen-binding site for MDR1, said first VH chain comprises an antigen-binding site for MDR1, said second VH chain comprises an antigen-binding site for said TAA, and said second VH chain binds to said TAA when paired with one of said VL chains, wherein said bispecific antibody binds to cancer cells expressing both MDR1 and said TAA, but exhibits reduced binding to non-cancer cells expressing MDR1 and / or said TAA. Bispecific antibody molecule. 2. The antigen-binding sites of said two VL chains comprise the light chain CDR 1-3 (LCDR 1-3) of a VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 1) where X 1 is N, Q or S, the bispecific antibody molecule according to embodiment 1. 3. Said two VL chains have the following sequences: (i) DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 2); (ii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO: 3); or (iii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO: 4) The bispecific antibody molecule according to embodiment 2, comprising LCDR 1-3 of the VL chain having 4. (i) LCDR1 comprises the sequence RSSQSIVHSTGX 1 TYLE (SEQ ID NO: 5); (ii) LCDR2 comprises the sequence KISNRFS (SEQ ID NO: 6); (iii) LCDR 3 comprises the sequence FQASHFPRT (SEQ ID NO: 7), where X 1 is N, Q or S, The bispecific antibody molecule according to embodiment 2 or 3, 5. The bispecific antibody molecule according to any one of embodiments 1-4, wherein the two VL chains are humanized. 6. The bispecific antibody molecule according to any one of embodiments 2-5, wherein the two VL chains comprise the following sequence, or amino acids that are at least 90% identical to the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 8); DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO: 3); or DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO: 4). 7. The bispecific antibody molecule according to embodiment 6, wherein the two VL chains comprise the following sequence, or a sequence that is at least 90% identical to the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 8). 8. The antigen-binding site of the first VH chain is the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 9) and comprises heavy chain CDRs 1-3 (HCDR 1-3) of a VH chain having the sequence, where X 2 is N, Q or S; the bispecific antibody molecule according to any one of embodiments 1-7. 9. The bispecific antibody molecule according to any one of embodiments 1-8, wherein the antigen-binding site of the first VH chain comprises HCDR 1-3 of a VH chain having the following sequence: (i) EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 10), or (ii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 11), or (iii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 12). 10. (i) HCDR1 contains the sequence RYTMS (SEQ ID NO: 13); (ii) HCDR2 contains the sequence TISSGGG X 2 TYYPDSVKG (SEQ ID NO: 14); (iii) HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15); where X 2 is N, Q or S, the bispecific antibody molecule according to embodiment 8 or 9. 11. The bispecific antibody molecule according to any one of embodiments 1 to 10, wherein the first and / or second VH chain is humanized. 12. The bispecific antibody molecule according to any one of embodiments 8 to 11, wherein the first VH chain comprises the following amino acid sequence, or an amino acid that is at least 90% identical to the following amino acid sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 16); EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 11); or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 12). 13. (i) LCDR1 contains the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 26), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGNTYYPDSVKG (SEQ ID NO: 29), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (ii) LCDR1 contains the sequence RSSQSIVHSTGQTYLE (SEQ ID NO: 27), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGQTYYPDSVKG (SEQ ID NO: 30), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (iii) LCDR1 contains the sequence RSSQSIVHSTGSTYLE (SEQ ID NO: 28), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGSTYYPDSVKG (SEQ ID NO: 31), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (iv) LCDR1 contains the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 26), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGQTYYPDSVKG (SEQ ID NO: 30), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (v) LCDR1 contains the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 26), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGSTYYPDSVKG (SEQ ID NO: 31), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (vi) LCDR1 contains the sequence RSSQSIVHSTGQTYLE (SEQ ID NO: 27), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGNTYYPDSVKG (SEQ ID NO: 29), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (vii) LCDR1 contains the sequence RSSQSIVHSTGSTYLE (SEQ ID NO: 28), LCDR2 contains the sequence KISNRFS (SEQ ID NO: 6), LCDR3 contains the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 contains the sequence RYTMS (SEQ ID NO: 13), HCDR2 contains the sequence TISSGGGNTYYPDSVKG (SEQ ID NO: 29), HCDR3 contains the sequence YGAGDAWFAY (SEQ ID NO: 15), or (viii) LCDR1 comprises the sequence RSSQSIVHSTGSTYLE (SEQ ID NO: 28), LCDR2 comprises the sequence KISNRFS (SEQ ID NO: 6), LCDR3 comprises the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 comprises the sequence RYTMS (SEQ ID NO: 13), HCDR2 comprises the sequence TISSGGGQTYYPDSVKG (SEQ ID NO: 30), HCDR3 comprises the sequence YGAGDAWFAY (SEQ ID NO: 15) or (ix) LCDR1 comprises the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 26), LCDR2 comprises the sequence KISNRFS (SEQ ID NO: 6), LCDR3 comprises the sequence FQASHFPRT (SEQ ID NO: 7), HCDR1 comprises the sequence RYTMS (SEQ ID NO: 13), HCDR2 comprises the sequence TISSGGGQTYYPDSVKG (SEQ ID NO: 30), HCDR3 comprises the sequence YGAGDAWFAY (SEQ ID NO: 15), A bispecific antibody molecule according to any one of aspects 2 to 12. 14. The bispecific antibody molecule according to any one of aspects 1 to 13, wherein the second VH chain is derived from a monospecific antibody molecule that binds to the TAA, and the affinity of the bispecific antibody molecule for the TAA when paired with one of the light chains is at least 2-fold lower than the affinity of the monospecific antibody molecule from which the VH chain is derived for the TAA. 15. The bispecific antibody molecule according to any one of aspects 1 to 14, wherein the TAA is CD47. 16. The antigen-binding site of the second VH chain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 17) The bispecific antibody molecule according to embodiment 15, comprising HCDR 1-3 of a VH chain containing 17. The bispecific antibody molecule according to embodiment 15 or 16, wherein the second VH chain comprises HCDR1 containing the sequence NYNMH (SEQ ID NO: 18), HCDR2 containing the sequence TIYPGNDDTSYNQKFKD (SEQ ID NO: 19), and HCDR3 containing the sequence GGYRAMDY (SEQ ID NO: 20). 18. The bispecific antibody molecule according to embodiment 15 or 16, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 17), EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 21), EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 22), or QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO:). 19. The bispecific antibody molecule according to any one of embodiments 1-14, wherein the TAA is PD-L1. 20. The antigen-binding site of the second VH chain has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32) The bispecific antibody molecule according to embodiment 19, comprising HCDR 1-3 of the VH chain containing . 21. The bispecific antibody molecule according to embodiment 19 or 20, wherein the antigen-binding site of the second VH chain comprises HCDR1 containing the sequence DSWIH (SEQ ID NO: 33), HCDR2 containing the sequence WISPYGGSTYYADSVKG (SEQ ID NO: 34), and HCDR3 containing the sequence RHWPGGFDY (SEQ ID NO: 35). 22. The bispecific antibody molecule according to any one of embodiments 19-21, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32). 23. The bispecific antibody molecule according to any one of embodiments 1-14, wherein the TAA is EGFR. 24. The antigen-binding site of the second VH chain has the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36) The bispecific antibody molecule according to embodiment 23, comprising HCDR 1-3 of the VH chain containing . 25. The bispecific antibody molecule according to embodiment 23 or 24, wherein the antigen-binding site of the second VH chain comprises HCDR1 comprising the sequence SGDYYWS (SEQ ID NO: 37), HCDR2 comprising the sequence YIYYSGSTDYNPSLKS (SEQ ID NO: 38), and HCDR3 comprising the sequence VSIFGVGTFDY (SEQ ID NO: 39). 26. The bispecific antibody molecule according to embodiment 24 or 25, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36). 27. The antigen-binding site of the second VH chain comprises the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40) The bispecific antibody molecule according to embodiment 23, comprising HCDR 1-3 of the VH chain comprising the above sequence. 28. The bispecific antibody molecule according to embodiment 26 or 27, wherein the second VH chain comprises HCDR1 comprising the sequence NYGVH (SEQ ID NO: 41), HCDR2 comprising the sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 42), and HCDR3 comprising the sequence ALTYYDYEFAY (SEQ ID NO: 43). 29. The bispecific antibody molecule according to any one of embodiments 26-28, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40). 30. The antigen-binding sites of the two VL chains are the following sequences: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25) The bispecific antibody molecule according to embodiment 1, comprising light chain CDRs 1-3 (LCDR 1-3) of a VL chain having the following sequence: 31. (i) LCDR1 comprises the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 44); (ii) LCDR2 comprises the sequence KISRLEA (SEQ ID NO: 45); (iii) LCDR3 comprises the sequence FQGSHFPRT (SEQ ID NO: 46), The bispecific antibody molecule according to embodiment 30. 32. The bispecific antibody molecule according to embodiment 30 or 31, wherein the VL chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25). 33. The antigen-binding site of the first VH chain is the following sequence: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA The bispecific antibody molecule according to any one of aspects 30 to 32, comprising heavy chain CDRs 1 to 3 (HCDR 1 to 3) of a VH chain having 34. (i) HCDR1 comprises the sequence SYTMS; (ii) HCDR2 comprises the sequence TISSGGGNTYYPDSVKG; (iii) HCDR3 comprises the sequence YYRYEAWFAS, The bispecific antibody molecule according to aspect 33. 35. The bispecific antibody molecule according to any one of aspects 30 to 34, wherein the first VH chain comprises the following amino acid sequence, or an amino acid that is at least 90% identical to the following amino acid sequence: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA. 36. The antigen-binding site of the first VH chain has the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 9) comprising heavy chain CDRs 1 to 3 (HCDR 1 to 3) of a VH chain having 2 where X 37. (i) HCDR1 comprises the sequence RYTMS (SEQ ID NO: 13); (ii) HCDR2 comprises the sequence TISSGGG X2TYYPDSVKG (SEQ ID NO: 14); (iii) HCDR3 comprises the sequence YGAGDAWFAY (SEQ ID NO: 15); where X2 is N, Q, or S, the bispecific antibody molecule according to aspect 36. 38. The bispecific antibody molecule according to embodiment 36 or 37, wherein the first VH chain comprises the following amino acid sequence, or an amino acid that is at least 90% identical to the following amino acid sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 16); EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 11); or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 12). 39. The antigen-binding site of the first VH chain is the following sequence: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA. The bispecific antibody molecule according to any one of embodiments 1 to 7, comprising heavy chain CDRs 1-3 (HCDR 1-3) of a VH chain having the following sequence: 40. (i) HCDR1 comprises the sequence SYTMS; (ii) HCDR2 comprises the sequence TISSGGGNTYYPDSVKG; (iii) HCDR3 comprises the sequence YYRYEAWFAS; The bispecific antibody molecule according to embodiment 39. 41. The bispecific antibody molecule according to embodiment 39 or 40, wherein the first VH chain comprises the following sequence, or an amino acid that is at least 90% identical to the following sequence: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA。 42. The second VH chain is derived from a monospecific antibody molecule that binds to the TAA, and the affinity of the bispecific antibody molecule for the TAA when paired with one of the light chains is at least 2-fold lower than the affinity of the monospecific antibody molecule from which the VH chain is derived for the TAA. The bispecific antibody molecule according to any one of aspects 30 to 41. 43. The bispecific antibody molecule according to any one of aspects 30 to 42, wherein the TAA is CD47. 44. The second VH chain has the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 17) The bispecific antibody molecule according to aspect 43, comprising HCDR 1-3 of the VH chain containing the same. 45. The second VH chain comprises HCDR1 containing the sequence NYNMH (SEQ ID NO: 18), HCDR2 containing the sequence TIYPGNDDTSYNQKFKD (SEQ ID NO: 19), and HCDR3 containing the sequence GGYRAMDY (SEQ ID NO: 20). The bispecific antibody molecule according to aspect 44. 46. The second VH chain contains the following sequence, or an amino acid sequence that is at least 90% identical to the following sequence. The bispecific antibody molecule according to aspect 44 or 45: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 17), EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 21), EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 22), or QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 23). 47. The bispecific antibody molecule according to any one of aspects 30 to 42, wherein the TAA is PD-L1. 48. The second VH chain has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32) The bispecific antibody molecule according to aspect 47, comprising HCDR 1-3 of the VH chain containing the same. 49. The bispecific antibody molecule according to aspect 47 or 48, wherein the second VH chain comprises HCDR1 containing the sequence DSWIH (SEQ ID NO: 33), HCDR2 containing the sequence WISPYGGSTYYADSVKG (SEQ ID NO: 34), and HCDR3 containing the sequence RHWPGGFDY (SEQ ID NO: 35). 50. The bispecific antibody molecule according to aspect 48 or 49, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32).

[0316] 51. The bispecific antibody molecule according to any one of aspects 30 to 42, wherein the TAA is EGFR. 52. The antigen-binding site of the second VH chain is the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36) The bispecific antibody molecule according to aspect 51, comprising HCDR 1 to 3 of the VH chain containing the same. 53. The bispecific antibody molecule according to aspect 52, wherein the second VH chain comprises HCDR1 containing the sequence SGDYYWS (SEQ ID NO: 37), HCDR2 containing the sequence YIYYSGSTDYNPSLKS (SEQ ID NO: 38), and HCDR3 containing the sequence VSIFGVGTFDY (SEQ ID NO: 39). 54. The bispecific antibody molecule according to aspect 52 or 53, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36). 55. The antigen-binding site of the second VH chain is the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40) The bispecific antibody molecule according to embodiment 51, comprising HCDR 1-3 of the VH chain containing . 56. The bispecific antibody molecule according to embodiment 52, wherein the antigen-binding site of the second VH chain comprises HCDR1 containing the sequence NYGVH (SEQ ID NO: 41), HCDR2 containing the sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 42), and HCDR3 containing the sequence ALTYYDYEFAY (SEQ ID NO: 43). 57. The bispecific antibody molecule according to embodiment 52 or 53, wherein the second VH chain comprises the following amino acid sequence, or an amino acid sequence that is at least 90% identical to the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40). 58. The bispecific antibody molecule according to any one of embodiments 1-57, wherein the antibody has more than 2-fold affinity for cells expressing both MDR1 and TAA as compared to cells expressing either MDR1 or TAA. 59. The antibody can increase the sensitivity of cancer cells to treatment with chemotherapeutic agents, wherein the half-maximal inhibitory concentration (IC50) of the chemotherapeutic agent when co-administered with the antibody is the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO: 24) and a VH chain having; the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25) and a VL chain having The bispecific antibody molecule according to any one of aspects 1 to 58, which is at least 2-fold lower than the IC50 of the chemotherapeutic agent when co-administered with the anti-MDR1 antibody 15D3 containing 60. The bispecific antibody molecule according to aspect 59, wherein the cancer cells are NALM6 ADR cells and optionally the chemotherapeutic agent comprises paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide or nilotinib 61. The bispecific antibody molecule according to any one of aspects 1 to 60, wherein the antibody inhibits efflux by MDR1 when bound to cells expressing MDR1 62. The antibody has the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO: 24) and a VH chain having The following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25) and a VL chain having The bispecific antibody molecule according to any one of aspects 1 to 61, which binds to MDR1 with an affinity at least 2-fold lower than that of the anti-MDR1 antibody 15D3 containing 63. The bispecific antibody molecule according to any one of aspects 1 to 62, wherein the antibody comprises an Fc domain modified to reduce or abolish binding of the antibody to one or more Fcγ receptors A bispecific antibody molecule according to any one of aspects 1 to 63 for use in a method of treating cancer in a subject, said method comprising administering said antibody to said subject. 65. The bispecific antibody molecule according to aspect 64, wherein said method comprises administering said antibody in combination with at least one further active agent, said at least one further active agent comprising a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. 66. The bispecific antibody molecule according to aspect 65, wherein said at least one further active agent is a chemotherapeutic agent, and optionally, said chemotherapeutic agent is taxol, a vinca alkaloid, or an anthracycline. A chemotherapeutic agent for use in a method of treating cancer in a subject, said method comprising administering said chemotherapeutic agent to said subject in combination with an antibody according to any one of aspects 1 to 63, and optionally, said chemotherapeutic agent is taxol, a vinca alkaloid, or an anthracycline. 68. The bispecific antibody molecule for use according to aspect 67, wherein the subject to be treated is a subject having cancer determined to be resistant to said chemotherapeutic agent. 69. A method of treating a subject for cancer, said method comprising administering to said subject a therapeutically effective amount of a bispecific antibody molecule according to any one of aspects 1 to 63. 70. The method according to aspect 69, wherein said method comprises administering said bispecific antibody molecule in combination with at least one further active agent, said at least one further active agent comprising a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. 71. The method according to aspect 70, wherein said at least one further active agent is a chemotherapeutic agent, and optionally, said chemotherapeutic agent is taxol, a vinca alkaloid, or an anthracycline. 72. The method according to embodiment 71, wherein the subject to be treated is a subject having cancer determined to be resistant to treatment with the chemotherapeutic agent, and optionally, the chemotherapeutic agent comprises paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide or nilotinib. 73. The following sequences: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK comprising the light chain CDR (LCDR) of the VL chain having, wherein X 1 is N, Q or S, a variable light (VL) chain, and the following sequences: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS comprising the heavy chain CDR (HCDR) of the VH chain having, wherein X 2 is N, Q or S, a variable heavy (VH) chain and comprising, or the following sequences: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25) a variable light (VL) chain comprising the light chain CDR (LCDR) of the VL chain having, and the following sequences: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA A variable heavy (VH) chain comprising a heavy chain CDR (HCDR) of a VH chain having comprising an antibody. 74. The VL chain has the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK and comprises LCDR 1-3 of the VL chain having The VH chain has the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS and comprises HCDR 1-3 of the VH chain having, or The VL chain has the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK and comprises LCDR 1-3 of the VL chain having The VH chain has the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS and comprises HCDR 1-3 of the VH chain having, or The VL chain has the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK comprising LCDR 1-3 of the VL chain having; said VH chain has the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS. comprising LCDR 1-3 of the VH chain having, the antibody according to embodiment 73. 75. Said LCDR1 comprises the sequence RSSQSIVHSTGNTYLE, RSSQSIVHSTGQTYLE, or RSSQSIVHSTGSTYLE, said LCDR2 comprises the sequence KISNRFS, said LCDR3 comprises the sequence FQASHFPRT, the antibody according to embodiment 74. 76. (i) Said HCDR1 comprises the sequence RYTMS; (ii) Said HCDR2 comprises the sequence TISSGGG X 2 TYYPDSVKG; (iii) Said HCDR3 comprises the sequence YGAGDAWFAY; where X 2 is N, Q or S, the antibody according to embodiment 75. 77. (i) LCDR1 comprises the sequence RSSQSIVHSTGNTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 comprises the sequence TISSGGGNTYYPDSVKG, HCDR3 comprises the sequence YGAGDAWFAY or, (ii) LCDR1 comprises the sequence RSSQSIVHSTGQTYLE, LCDR2 comprises the sequence KISNRFS, LCDR3 comprises the sequence FQASHFPRT, HCDR1 comprises the sequence RYTMS, HCDR2 contains the sequence TISSGGGQTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (iii) LCDR1 contains the sequence RSSQSIVHSTGSTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGSTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (iv) LCDR1 contains the sequence RSSQSIVHSTGNTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGQTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (v) LCDR1 contains the sequence RSSQSIVHSTGNTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGSTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (vi) LCDR1 contains the sequence RSSQSIVHSTGQTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGNTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (vii) LCDR1 contains the sequence RSSQSIVHSTGSTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGNTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (viii) LCDR1 contains the sequence RSSQSIVHSTGSTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGQTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, or (ix) LCDR1 contains the sequence RSSQSIVHSTGNTYLE, LCDR2 contains the sequence KISNRFS, LCDR3 contains the sequence FQASHFPRT, HCDR1 contains the sequence RYTMS, HCDR2 contains the sequence TISSGGGQTYYPDSVKG, HCDR3 contains the sequence YGAGDAWFAY, The antibody according to embodiment 74 or 75. 78. The VL chain of the antibody contains the following amino acid sequence, or contains an amino acid sequence that is at least 90% identical to the following amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX1TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK The VH chain of the antibody contains the following amino acid sequence, or contains an amino acid sequence that is at least 90% identical to the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX2TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS The antibody according to any one of aspects 73 to 77. 79. The antibody, wherein: it comprises a variable light (VL) chain containing light chain CDRs 1 to 3 (LCDRs) of the VL chain having the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25) and a variable heavy (VH) chain containing heavy chain CDRs 1 to 3 (HCDRs) of the VH chain having the following sequence: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA and is the antibody according to aspect 73. 80. (i) LCDR1 comprises the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 44); (ii) LCDR2 comprises the sequence KISRLEA (SEQ ID NO: 45); (iii) LCDR3 comprises the sequence FQGSHFPRT (SEQ ID NO: 46); (i) HCDR1 comprises the sequence SYTMS; (ii) HCDR2 comprises the sequence TISSGGGNTYYPDSVKG; (iii) HCDR3 comprises the sequence YYRYEAWFAS, and is the antibody according to aspect 79. 81. The VL chain of the antibody comprises, or comprises an amino acid sequence that is at least 90% identical to, the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25); The VH chain of the antibody comprises the following amino acid sequence or an amino acid sequence that is at least 90% identical to the following amino acid sequence: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA; The antibody according to embodiment 79 or 80. 82. The antibody according to any one of embodiments 73 to 81, wherein the antibody is a monospecific bivalent antibody that specifically binds to MDR-1. 83. The antibody according to any one of embodiments 73 to 81, wherein the antibody is a bispecific antibody that comprises the VL chain as a common light chain. 84. The antibody according to embodiment 83, wherein the bispecific antibody comprises an MDR-1 binding domain and a tumor-associated antigen (TAA) binding domain, and wherein each of the MDR-1 binding domain and the TAA binding domain comprises LCDR 1-3 of the VL chain. 85. The TAA is CD47, and the TAA binding domain has the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS The antibody according to embodiment 84, which comprises HCDR 1-3 of the VH chain having the above sequence. 86. The VH chain of the CD47 binding domain comprises HCDR1 comprising the sequence NYNMH, HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD, and HCDR3 comprising the sequence GGYRAMDY. The antibody according to embodiment 85. 87. The TAA is PD-L1, and the PD-L1 binding domain is the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32) The antibody according to embodiment 84, comprising HCDR 1-3 of the VH chain having the above. 88. The VH chain of the PD-L1 binding domain comprises HCDR1 comprising the sequence DSWIH (SEQ ID NO: 33), HCDR2 comprising the sequence WISPYGGSTYYADSVKG (SEQ ID NO: 34), and HCDR3 comprising the sequence RHWPGGFDY (SEQ ID NO: 35). The antibody according to embodiment 87. 89. The TAA is EGFR, and the EGFR binding domain is the following sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36) The antibody according to embodiment 84, comprising the HCDR of the VH chain having the above. 90. The VH chain of the EGFR binding domain comprises HCDR1 comprising the sequence SGDYYWS (SEQ ID NO: 37), HCDR2 comprising the sequence YIYYSGSTDYNPSLKS (SEQ ID NO: 38), and HCDR3 comprising the sequence VSIFGVGTFDY (SEQ ID NO: 39). The antibody according to embodiment 89. 91. The TAA is EGFR, and the EGFR binding domain is the following sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40) The antibody according to embodiment 84, comprising the HCDR of the VH chain having the above. 92. The antibody according to embodiment 91, wherein the VH chain of the EGFR binding domain comprises HCDR1 comprising the sequence NYGVH (SEQ ID NO: 41), HCDR2 comprising the sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 42), and HCDR3 comprising the sequence ALTYYDYEFAY (SEQ ID NO: 43). 93. The following sequences: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK having, wherein X 1 is N, Q or S, or the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 25) a variable light (VL) chain comprising a light chain CDR (LCDR) of the VL chain having; The following sequences: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS; or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32); or QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36); or ​QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40) A variable heavy (VH) chain comprising a heavy chain CDR (HCDR) of the VH chain having an antibody. 94. The VL chain is (i) an LCDR1 comprising the sequence RSSQSIVHSTGX 1 TYLE (SEQ ID NO: 5); (ii) an LCDR2 comprising the sequence KISNRFS (SEQ ID NO: 6); (iii) an LCDR3 comprising the sequence FQASHFPRT (SEQ ID NO: 7), where X 1 is N, Q or S, the VH chain is (i) an HCDR1 comprising the sequence NYNMH, an HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD, and an HCDR3 comprising the sequence GGYRAMDY; or (ii) an HCDR1 comprising the sequence DSWIH (SEQ ID NO: 33), an HCDR2 comprising the sequence WISPYGGSTYYADSVKG (SEQ ID NO: 34), and an HCDR3 comprising the sequence RHWPGGFDY (SEQ ID NO: 35); or (iii) an HCDR1 comprising the sequence SGDYYWS (SEQ ID NO: 37), an HCDR2 comprising the sequence YIYYSGSTDYNPSLKS (SEQ ID NO: 38), and an HCDR3 comprising the sequence VSIFGVGTFDY (SEQ ID NO: 39); or (iv) an HCDR1 comprising the sequence NYGVH (SEQ ID NO: 41), an HCDR2 comprising the sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 42), and an HCDR3 comprising the sequence ALTYYDYEFAY (SEQ ID NO: 43) The antibody according to embodiment 93, comprising 95. The VL chain comprises (i) an LCDR1 comprising the sequence RSSQSIVHSTGNTYLE (SEQ ID NO: 44); (ii) an LCDR2 comprising the sequence KISRLEA (SEQ ID NO: 45); (iii) an LCDR3 comprising the sequence FQGSHFPRT (SEQ ID NO: 46); wherein the VH chain is HCDR1 containing the sequence NYNMH, HCDR2 containing the sequence TIYPGNDDTSYNQKFKD, and HCDR3 containing the sequence GGYRAMDY; or HCDR1 containing the sequence DSWIH (SEQ ID NO: 33), HCDR2 containing the sequence WISPYGGSTYYADSVKG (SEQ ID NO: 34), and HCDR3 containing the sequence RHWPGGFDY (SEQ ID NO: 35); or HCDR1 containing the sequence SGDYYWS (SEQ ID NO: 37), HCDR2 containing the sequence YIYYSGSTDYNPSLKS (SEQ ID NO: 38), and HCDR3 containing the sequence VSIFGVGTFDY (SEQ ID NO: 39); or HCDR1 containing the sequence NYGVH (SEQ ID NO: 41), HCDR2 containing the sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 42), and HCDR3 containing the sequence ALTYYDYEFAY (SEQ ID NO: 43) The antibody according to embodiment 93, comprising 96. The antibody according to any one of embodiments 93 to 95, wherein the antibody is a bispecific antibody. 97. The antibody according to any one of embodiments 1 to 96, wherein the antibody is a humanized antibody or a chimeric antibody containing a human Fc domain. 98. The antibody according to embodiment 97, comprising an immunoglobulin G1 (IgG1) Fc domain. 99. The antibody according to any one of embodiments 1 to 98, wherein the first VH chain is fused to the first Fc domain and the second VH chain is fused to the second Fc domain. 100. The antibody according to embodiment 99, wherein the Fc domain comprises a modified CH3 domain that preferentially forms a heterodimer containing the first and second VH chains.

[0317] 101. The antibody according to embodiment 100, wherein the first and second Fc domains are human immunoglobulin G1 (IgG1) Fc domains. 102. The antibody according to any one of embodiments 1 to 101, and a pharmaceutically acceptable excipient A pharmaceutical composition comprising 103. The pharmaceutical composition according to embodiment 102, further comprising at least one additional active agent. 104. The pharmaceutical composition according to embodiment 103, wherein the at least one additional active agent comprises a chemotherapeutic agent. 105. The pharmaceutical composition according to embodiment 104, wherein the chemotherapeutic agent is paclitaxel, a vinca alkaloid, or an anthracycline. 106. The pharmaceutical composition according to any one of embodiments 103 to 105, wherein the at least one additional active agent comprises an inhibitor of a multidrug resistance transporter. 107. The pharmaceutical composition according to embodiment 103, wherein the at least one additional active agent comprises an immunotherapeutic agent. 108. One or more nucleic acids comprising one or more sequences encoding the antibody according to any of the above embodiments. 109. The one or more nucleic acids according to embodiment 108, wherein the one or more sequences are operably linked to a promoter. 110. One or more recombinant expression vectors comprising the one or more nucleic acids according to embodiment 108 or 109. 111. Mammalian cells genetically engineered with the one or more recombinant expression vectors according to embodiment 110. 112. The cells according to embodiment 111, wherein the cells are immune cells. 113. An antibody according to any of embodiments 1 to 63 and 73 to 101 or a nucleic acid encoding the same, and at least one additional active agent A kit comprising 114. The kit according to embodiment 113, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. 115. A method of killing cancer cells, the method comprising contacting the cancer cells with an antibody according to any one of embodiments 1 to 63 and 73 to 101. 116. The method according to embodiment 115, further comprising administering at least one additional active agent. 117. The method according to embodiment 116, wherein the at least one additional active agent comprises a chemotherapeutic agent. 118. The method according to embodiment 116 or 117, wherein the method increases the killing of the cancer cells by at least 5% as compared to contacting with the at least one additional active agent alone. 119. The method according to any one of embodiments 115 - 118, wherein the cancer cells are drug - resistant cancer cells. 120. A method for treating a subject for cancer, the method comprising administering to the subject the antibody according to any one of embodiments 1 - 63 and 73 - 101 or the pharmaceutical composition according to any one of embodiments 102 - 107. 121. The method according to embodiment 120, wherein the subject is a subject previously treated for the cancer. 122. The method according to embodiment 120 or 121, wherein the cancer is drug - resistant or multi - drug resistant. 123. The method according to embodiment 122, wherein the cancer is resistant to chemotherapeutic agents. 124. The method according to embodiment 122, wherein the cancer is resistant to immunotherapeutic agents. 125. The method according to any one of embodiments 120 - 122, wherein the cancer is resistant to inhibitors of multi - drug resistance transporters. 126. The method according to any one of embodiments 120 - 125, further comprising administering to the subject at least one additional active agent. 127. The method according to embodiment 126, wherein the at least one additional active agent comprises a chemotherapeutic agent. 128. The method according to embodiment 127, wherein the chemotherapeutic agent is taxol, a vinca alkaloid, or an anthracycline. 129. The method according to any one of embodiments 126 - 128, wherein the at least one additional active agent comprises an inhibitor of multi - drug resistance transporters. 130. The method according to any one of embodiments 126 - 128, wherein the at least one additional active agent comprises an immunotherapeutic agent. 131. The method according to any one of aspects 126 - 128, wherein the method increases the effectiveness of the at least one additional active agent as compared to treatment with the at least one additional active agent alone. 132. The method according to aspect 131, wherein the increased effectiveness comprises at least a 5% increase in cancer cell killing. 133. The method according to any one of aspects 120 - 132, further comprising analyzing a sample of the cancer to determine whether the cancer expresses MDR1 above a predetermined threshold, a tumor - associated antigen (TAA) above a predetermined threshold, or both, wherein optionally the TAA comprises CD47. 134. The method according to aspect 133, wherein the predetermined threshold corresponds to the level of MDR1 and / or TAA expressed by reference cells. 135. The method according to aspect 134, wherein MDR1 and / or TAA in the reference cells are knocked out or knocked down. 136. The method according to aspect 134 or 135, wherein the reference cells are non - cancerous cells. 137. The method according to aspect 134, wherein the non - cancerous cells express normal levels of MDR1 and / or TAA. 138. When the cancer expresses MDR1 and TAA above a predetermined threshold, administering the multispecific antibody to the subject, and when the cancer expresses MDR1 or TAA below the predetermined threshold, performing conventional treatment without administering the multispecific antibody to the subject. The method according to any one of aspects 120 - 137. 139. A method of making a multispecific antibody that specifically binds to cells expressing both multidrug resistance protein 1 (MDR1) and tumor - associated antigen (TAA), wherein optionally the TAA comprises CD47, PD - L1, or EGFR, and the method comprises: producing a multispecific antibody comprising an MDR1 - binding domain and a TAA - binding domain; contacting a first cell expressing MDR1 and TAA with the multispecific antibody; contacting a second cell expressing either MDR1 or TAA with the multispecific antibody; Determining a binding specificity ratio by comparing the binding of the multispecific antibody to the first cell with the binding of the multispecific antibody to the second cell; and Identifying the multispecific antibody as specific for cells expressing both MDR1 and TAA when the ratio exceeds a predetermined threshold A method comprising the steps of: 140. The method according to embodiment 139, wherein the predetermined threshold is greater than 2:1. 141. The method according to embodiment 139 or 140, wherein the second cell expresses MDR1 but does not express TAA, and the method further comprises contacting a third cell that expresses TAA but does not express MDR1 with the multispecific antibody. 142. The method according to embodiment 139 or 140, wherein the method further comprises contacting the first cell, the second cell, and / or the third cell with a control antibody selected from a monospecific anti-MDR1 antibody and a monospecific anti-TAA antibody. 143. A recombinant human cell line comprising an exogenous nucleic acid encoding a multi-drug resistance protein 1 (MDR1) due to overexpression of MDR1, wherein the cell line expresses a tumor-associated antigen (TAA), and optionally the TAA comprises the leukocyte surface antigen CD47. 144. The cell line according to embodiment 143, wherein the cell line is a kidney cell line. 145. The cell line according to embodiment 144, wherein the cell line is a HEK 293T cell line. 146. A method for producing the cell line according to any one of embodiments 143 to 145, comprising: Contacting a human cell expressing the TAA with the exogenous nucleic acid under conditions sufficient to introduce the exogenous nucleic acid into the cell to produce a recombinant human cell that expresses the TAA and stably overexpresses MDR1; and Culturing the recombinant human cell under conditions sufficient to produce a recombinant human cell line that expresses the TAA and stably overexpresses MDR1 A method comprising the steps of:

[0318] The following examples are provided by way of illustration and not limitation.

Example

[0319] The following examples are presented to provide a complete disclosure and description of how to make and use the present invention to those skilled in the art, and are not intended to limit the scope that the inventors regard as the invention, nor are they intended to represent all or the only experiments in which the following experiments were conducted. Although efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0320] General methods in molecular and cell biochemistry are described in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for the methods referred to or related to in the present disclosure are available from commercial companies such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., as well as repositories such as Addgene, Inc., American Type Culture Collection (ATCC).

[0321] [Example 1: Generation of a bispecific mAb that specifically binds to and re-sensitizes cells co-expressing Pgp (MDR1) and CD47, but does not bind to cells with reduced or absent expression of either protein] This example demonstrates the development of an antibody-format-based molecule that can effectively block efflux by binding to the extracellular domain of EP in a selective manner while specifically targeting antigens expressed by cancer cells. EP blockade results in the resensitization and killing of cells that are resistant or have become resistant to chemotherapeutic agents. In this example, a bispecific antibody molecule was constructed that binds to the extracellular domain (ECD) of EP and blocks efflux while targeting a cancer immune checkpoint protein. The application of the bispecific antibody described below resensitizes cells to chemotherapeutic agents in a selective manner when EP is co-expressed with the target immune checkpoint protein.

[0322] Materials and Methods

[0323] [Cell Lines and Cell Survival Assays] The HEK 293T, MCF-7, N6ADR, and SKNF7 cell lines expressing Pgp were obtained from the American Type Culture Collection. The N6 / ADR cells are also called NALM6 / ADR cells. All cell lines and their derived strains were maintained in RPMI 1640 or DMEM supplemented with up to 10% fetal bovine serum (Sigma), non-essential amino acids, and 2 mmol / L L-glutamine in a humidified incubator at 37 °C and 5% CO2 (unless otherwise indicated). Cells were used as supplied or were modified to overexpress (Ox) Pgp or had Pgp expression knocked down (KD) by lentivirus-mediated short hairpin RNA or had the functional CD47 gene knocked out (KO) by CRISPR / Cas-mediated knockout technology as essentially described (Cong, L. et al. (2013) Science 339, 819-823). To measure the IC50 of vincristine and paclitaxel, cells were plated in normal medium and allowed to adhere overnight. Paclitaxel or vincristine (Sigma) was added in dilution series, and any modulator was added in the range of 0 to 500 μM / L. After 72 hours, cell viability was measured using the Celltiter-Glo luminescent cell viability assay (Promega). The drug concentration (IC50) that produced 50% inhibition of cell viability was calculated from multi-parameter curve fitting (GraphPad Prism software, GraphPad Software, Inc.) and determined from at least two replicate experiments. In most of the experiments conducted, cell lines that did not show a 50% decrease in cell viability in response to drug and / or modulator treatment were considered to have not reached the IC50 by definition and were listed as having an IC50 > 1000 nmol / L for the paclitaxel or drug / modulator combination under study.

[0324] Recombinant cell lines stably expressing the described monoclonal antibody (mAb) were also generated.

[0325] [Recombinant DNA technology] As described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, standard methods were used to manipulate DNA. Reagents were used according to the manufacturer's instructions. General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is described in Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242. The amino acids of the antibody chains are numbered and referred to according to Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., (1991).

[0326] [DNA sequencing] The DNA sequence was determined by double-stranded sequencing.

[0327] [Sequence analysis and sequence data management of DNA and proteins] The Vector NTI (ThermoFisher) software package was used for sequence mapping, analysis, annotation, and illustration.

[0328] [Cell culture techniques and antibody production] Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.

[0329] mAbs, Fab’2, Fabs, and bispecific mAbs were transiently produced using 293 and CHO cells. Different antibody constructs were expressed using polymer-based co-transfection of Expi293 cells (A14527, ThermoFisher). Cells with mammalian expression vectors were cultured in suspension according to the manufacturer's recommendations.

[0330] To prepare bispecific constructs, cells were transfected with the corresponding expression vectors at a 1:1:4 ratio (heavy chain KK: heavy chain DD: light chain). A 1:2 ratio (H chain: L chain) was used for standard antibody expression.

[0331] Six days after transfection, cells were harvested by centrifugation. Specifically, a total of 1 μg of coding DNA per 1 ml of transfected culture was diluted in OptiMEM® medium (Life Technologies) and incubated with Expifectamine reagent (Life Technologies) in the same medium for 20 minutes. This mixture was then added to Expi293® cells growing in suspension in Expi 293® expression medium (Life Technologies) at 250 million cells / ml and 37°C with 8% CO2 in air. Six days later, the medium containing the antibody construct was harvested by centrifugation.

[0332] [Reagents and cell lines used to test binding, efflux blockade, and cell sensitization to chemotherapeutic agents] Human embryonic kidney (HEK) cell line HEK 293 FT (Life Technologies) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Life Technologies), 100 U / mL penicillin, and 100 μg / mL streptomycin, with incubation at 37 °C in 5% CO2.

[0333] 293T cells were transiently transfected with a human P-glycoprotein tagged ORF clone in the pLentic-C-Myc-DDK-P2A-Puro plasmid using the optimized PEIPro™ transfection protocol (Polyplus). DNA and JetPEI® were diluted in the culture medium and gently mixed for approximately 10 minutes. This mixing formed the transfection complex, which was added directly to the cell culture. Efflux blockade was measured using the Multidrug Resistance Direct Dye Efflux assay (Chemicon) according to the manufacturer's protocol.

[0334] [Sources of target sequence, antibody sequence, and specific anti-target antibody sequence] The human tagged ORF clone of P-glycoprotein (Pgp), also known as multidrug resistance protein 1 (MDR1) (gene ABCB1) (NM_000927) in pLenti-C-Myc-DDK-P2A-Puro was obtained from Origene, the CD47 antibody (CC2C6, Seiffert M, et al. (1999) Blood 94:3633) was obtained from Biolegend, and the anti-ABCB1 JSB-1 (MAB4120) was obtained from Millipore.

[0335] [Generation of stable ABCB1 overexpressing (Ox) cell lines] To characterize both the binding and in vitro potency, a cell line stably overexpressing ABCB1 was developed. Adherent 293T naive cells obtained from the American Type Culture Collection (ATCC) were utilized. This cell line endogenously expresses ABCB1 at low to moderate levels on the cell surface, as characterized by flow cytometry using a commercially available ABCB1 antibody (BioLegend, clone 4E3.16). 293T naive cells were transfected with ABCB1 using the Polyplus PEIpro reagent. Three days after transfection, the cells were placed under selection using a hygromycin B solution (Millipore Sigma). Fourteen days after continuous hygromycin B selection, 293T cells were evaluated for ABCB1 cell surface expression. To ensure that non-transfected cells did not expand in subsequent cultures, bulk sorting using fluorescence-activated cell sorting (FACS) of ABCB1-positive 293T cells was performed using a FACSAriaI (BD Biosciences). The bulk-sorted 293T ABCB1 overexpressing cells were expanded and subsequently reconfirmed for ABCB1 overexpression. Using a similar method, ABCB1 overexpressing cells were generated from 293T-CD47 knockout cells generated as described herein.

[0336] [Generation of a stable ABCB1 KD 293T cell line] To characterize both the binding and in vitro potency, a cell line with stable knockdown of ABCB1 expression was developed. Lentivirus was produced in 293T naive cells by transfection using the R8.74 helper plasmid, the VSVG envelope plasmid, and the GE Dharmacon GIPZ lentiviral vector containing shRNA against ABCB1. The harvested lentivirus was then used to transduce adherent 293T naive cells. Three days after transduction, 293T transduced cells were evaluated for ABCB1 cell surface expression by flow cytometry (BioLegend, clone 4E3.16). Transduced 293T cells did not show ABCB1 expression compared to 293T naive cells that endogenously express ABCB1 at low levels. In addition, the GIPZ lentiviral vector contains GFP. All transduced 293T cells were GFP+, indicating successful transduction along with decreased expression. The lack of ABCB1 expression was reconfirmed by flow cytometry in subsequent passages.

[0337] [Generation of KO cell lines] To construct knockout HEK293 cell lines for the ABCB1 and CD47 genes, HEK293 host cells were first cultured via adherent culture in DMEM (Dulbecco's Modified Eagle's Medium Gibco, Grand Island, N.Y., USA) supplemented with 10% (v / v) FBS and glutamine. Cells were cultured at 5% CO2, 37 °C with saturated humidity.

[0338] The design of gRNAs was performed using the online CHOPCHOP web tool (see Kornel Labun et al., (2016). Nucleic Acids Research; and Tessa G. Montague et at., (2014) Nucleic Acids Res. 42:W401-W407) to select target sites for CRISPR / Cas9, CRISPR / Cpf1 or TALEN-induced mutagenesis. All designed gRNAs were chemically synthesized (ThermoFisher).

[0339] Transfection of 293T cells was performed by lipid-based transfection using CRISPRMax reagent (ThermoFisher) according to the manufacturer's protocol. Briefly, one day before transfection, adherent cells were plated at 0.2×10 5 cells / well on a 96-well plate. On the day of transfection, solutions of GeneArt Platinum Cas9 protein, gRNA and transfection reagent were added to the cells. Seventy-two hours after transfection, single cells were selected by limiting dilution and then cell culture was continued in a 96-well plate format for two weeks. Next, the selected clones were passaged to a 24-well plate and tested by genotyping using the Guide-it kit (Takara) according to the manufacturer's protocol. The genomic region surrounding the CRISPR target site of each gene was PCR amplified to determine whether gene editing had generated indels on one allele (single allele) or both alleles (both alleles) in single isolated clones. The expression of the target protein on clones with mutations in both alleles was tested by FACS.

[0340] [Construction of the human-mouse sequence of the molecule tested (human Fc, mouse Fv)] Expression vector: For the preparation of the antibody expression vector, the variable regions of the heavy and light chain DNA sequences were subcloned in-frame into either the human IgG1 constant heavy chain or the human IgG1 kappa constant light chain, which had been pre-inserted into their respective general recipient expression vectors optimized for expression in mammalian cell lines. The gene to be expressed was cloned into the pCI-neo mammalian expression vector (Promega) using the full-length human cytomegalovirus (CMV) immediate early promoter for high-level gene expression. The two antibody chains were cloned into two different vectors.

[0341] The N-terminal signal sequences from the mouse IgG heavy and kappa light chains were used for the secretory expression of the heavy and light chains, respectively. The signal peptide was cleaved during expression, leaving the intact N-terminus. In the Fab construct, the C-terminus of the CH1 IgG1 constant region was fused to a 6×His tag to facilitate purification.

[0342] For the generation of bispecific antibody vectors, the bispecific molecule derived from IgG1 contains at least two antigen-binding portions capable of specifically binding to two different targets, namely Pgp (ABCB1) and CD47. The antigen-binding portions are Fab fragments composed of a heavy chain and a light chain, each containing a variable region and a constant region. A common light chain was identified that could pair and provide a binding acceptable as both Fab anti-Pgp (aPgp) and Fab anti-CD47 (aCD47); the use thereof enabled avoidance of LC mispairing (see, for example, U.S. Patent No. 8,765,412, the entire disclosure of which is incorporated herein by reference). A bispecific construct was made based on the electrostatic steering effect (see, for example, Gunasekeran et al., (2010) Journal of Biological Chemistry 285, 19637-19646; the disclosure thereof is incorporated herein by reference in its entirety). Briefly, the polypeptide chain or half-antibody against the target is assembled as a bispecific antibody via charge substitution in the CH3 domain: one heavy chain contains K392D and K409D substitutions, and the other contains E356K and D399K substitutions.

[0343] Figure 2 provides a schematic of the bispecific mAb, which contains sections both containing sequences from A, 15D3; B, 5F9 on human IgG1Fc, together with C, a common light chain kappa sequence from MRK16.

[0344] The previously produced monoclonal antibodies 15D3 (see, for example, U.S. Patent No. 5,959,084; the disclosure thereof is incorporated herein by reference in its entirety) and MRK16 (Iwahashi et al., Cancer Research 53, 1993; the disclosure thereof is incorporated herein by reference in its entirety) against Pgp were cloned as recombinant engineered antibodies into a human IgG1 / kappa expression vector.

[0345] Variable heavy and light chain fragments from the mouse hybridoma array were available and cloned into the same background of the leader and constant regions.

[0346] The variable heavy and light fragments of an anti-CD47 (5F9; see, e.g., U.S. Patent No. 9,017,675; the disclosure of which is incorporated herein by reference in its entirety) antibody were cloned into the same background of the leader and constant regions in two separate vectors.

[0347] Numerous combinations of these and other heavy and light chain fragments were made and tested. Most of the constructs tested did not yield acceptable binding or activity. Examples of the antibodies constructed and tested, as well as their binding and cell killing properties, are provided in FIG. 8.

[0348] FIG. 8 provides a comparison of binding and enhanced cell killing by different bispecific antibodies (having various combinations of humanized or chimerized heavy or light chains). Specifically, column 1 of FIG. 8 (“Ab”) provides the various bispecific formats tested. For example, in row 1, 15D3 IgG1 DD / 5F9 IgG1 KK / MRK16 shows i) a 15D3 heavy chain sequence having the DD CH3 mutation described herein (chimerized in a human IgG1 framework), ii) a 5F9 heavy chain sequence having the KK CH3 mutation described herein, and iii) an MRK16 light chain (chimerized in a human IgG kappa framework). All light chains shown were of the human kappa type. Binding (FIG. 8, columns 2-3; “Kd (nM)” and “Bmax”) and killing (FIG. 8, column 3; “killing”) were determined as follows: “Kd (nM)”, binding by ELISA (solid phase having Fc-tagged CD47 on the plate as the capture layer); “Bmax”, Bmax binding to the plate described for “Kd (nM)”; “Killing”, paclitaxel titration (20 μM - 10 -8Enhanced 293 naive cell killing (sensitization) in the presence of [[ID=]], μM range); "-" = zero, "±" = 0 to 0.5 Log shift (at 50% cell killing curve): "+" = 1 to 2 Log shifts, "++" ≥ 2 Log shifts. Columns 5 - 8 ("Binding (293)", "Binding (293 KO CD47)", "Binding (293 KO CD47, OX ABCB1)", and "Binding (293 OX ABCB1)") provide the FACS mean fluorescence intensity of antibodies binding to the indicated cell lines compared to an unbound control antibody: "-" = ~0, "±" = Log shift from 0 to ~0.5, "+" = ~1 Log shift, "++" = 1 to >2 Log shifts (see Figure 3A for examples of unbound control and binding antibodies).

[0349] Of the antibodies constructed and tested, those showing unacceptable binding properties or inefficiency in killing target cells expressing both MDR1 and CD47 (especially compared to lead candidates) are worth mentioning.

[0350] For example, as shown in Figure 8, when an antibody containing the heavy chain charge exchange Fc regions of 15D3 and 5F9 and the common MRK16-derived light chain (referred to as "15D3 IgG1 DD / 5F9 IgG1 KK / MRK16") was tested, it exhibited the desired binding properties and resulted in enhanced killing (++) of target cells expressing both MDR1 and CD47. Therefore, this antibody was selected as a lead candidate. In comparison, when the common light chain of the lead candidate (i.e., "MRK16") was replaced with an alternative common light chain, such as in the case of "UIC2" (see, for example, "15D3 IgG1 DD / 5F9 IgG1 KK / UIC2") or "9F11" (see, for example, "15D3 IgG1 KK / 5F9 IgG1 DD / 9F11"), specific killing of cells expressing both CD47 and MDR1 was essentially not observed. In addition, replacing the "15D3" heavy chain with an alternative anti-MDR1 heavy chain, such as "MM4.17 2" or "UIC2" heavy chain, also significantly reduced the specific killing of cells expressing both CD47 and MDR1 (see, for example, "MM4.17 2 IgG1 DD / 5F9 IgG1 KK / MRK16" and "UIC2 IgG1 KK / 5F9 IgG1 DD / MRK16" in Figure 8).

[0351] The effect of exchanging the light chain used (i.e., "LC shuffling") on antibodies that bind to cells expressing an antigen was evaluated by ELISA and FACS binding assays. In tests of LC shuffling using a CD47-specific heavy chain, the modified anti-CD47 heavy chain (5F9) was combined with the M89, 15D3, MRK16, or UIC2 light chain, and binding was evaluated. Results showing the effect of LC shuffling on the binding of bispecific antibodies of various anti-MDR1 molecules to CD47-Fc coated plates by ELISA are provided in Table 2.

[0352] [Table 2]

[0353] Of note in the table above is the 100-fold decrease in affinity seen with the 5F9 / MRK16 combination compared to the non-swapped 5F9 control. Antibodies having a Kd similar to that of the 5F9 / MRK16 combination for CD47 are preferred, as they provide the degree of specificity necessary when used in a bispecific format such that the bispecific antibody has lower binding to normal cells and increased specificity for cancer cells that express CD47 at higher levels than normal cells.

[0354] Table 3 provides the results of an LC shuffle test showing the effect on CD47 binding of combining the 15D3 heavy chain with the UIC2, MRK16, or 5F9 light chain, measured by ELISA.

[0355]

Table 3

[0356] Of note in the table above is the surprising affinity of 15D3 for CD47, which decreased by approximately 20-fold after combination of 15D3 HC and MRK16 LC compared to the non-swapped 15D3 control.

[0357] Candidate constructs having combinations of 15D3 and 5F9 heavy chains with the common MRK16 light chain have acceptable binding and target antagonizing properties. In comparison, other combinations of heavy and light chains tested showed unacceptable binding and / or target cell killing results when tested. See Figure 8.

[0358] [Purification of mAb] To purify antibody formats containing Fc, 10 μl of MabSelect™ SuRe™ (GE Healthcare) per 1 ml of supernatant was added to the harvested medium and stirring was continued overnight at 4°C. The next day, protein A resin was applied to a 24-well filter plate using a vacuum manifold unit (Pall Life Sciences, USA). The resin was washed with PBS and the antibody was eluted with 50 mM phosphoric acid pH 3 and neutralized with 10× PBS pH 13.

[0359] The histidine-tagged Fab was purified according to the same procedure using Ni Sepharose 6 Fast Flow histidine-tagged protein purification resin (GE Healthcare). The beads were washed with PBS and then with 25 mM phosphate buffer pH 7.4, 150 mM NaCl supplemented with 20 mM imidazole. The complex was eluted with two volumes of 25 mM phosphate buffer pH 7.4, 150 mM NaCl supplemented with 500 mM imidazole. Finally, the purified Fab was buffer-exchanged into PBS.

[0360] Array

[0361] Pgp / MDR1 has the following amino acid sequence.

[0362] Nucleic acid sequences encoding Pgp / MDR1 are available: P-glycoprotein (ABCB1) (NM_000927), Homo sapiens genomic DNA, ATP-binding cassette subfamily B member 1 (ABCB1), RefSeq gene on chromosome 7 (NG_011513 gen)

[0363] Nucleic acid sequences encoding CD47 are available: Human CD47 molecule (CD47), transcript variant 1, mRNA NCBI reference sequence: NM_001777.3

[0364] The variable heavy chain sequence of the anti-CD47 5F9 antibody is as follows. QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0365] The variable light chain sequence of the anti-CD47 5F9 antibody is as follows. DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADVGVYYCFQGSHVPYTFGQGTKLEIK.

[0366] The variable heavy chain sequence of the anti-MDR1 MRK16 antibody is as follows. EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA.

[0367] The sequence of the variable light chain of the MRK16 antibody is as follows. DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK.

[0368] The sequence of antibody 15D3 is available from U.S. Patent No. 5,849,877, where the heavy chain sequence of antibody 15D3 is as follows. EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA And the light chain sequence of antibody 15D3 is as follows. DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK.

[0369] The light chain sequence of the anti-MDR1 UIC2 antibody as used as described herein is as follows. DVVMTQTPRSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHIPPWTFGGGTKLDIK.

[0370] The heavy chain sequence of the anti-MDR1 UIC2 antibody as used as described herein is as follows. AVQLQQSGPELVKTGASVKISCKASGYSFSNYYIHWVKQSHGKSLEWIGFISCYNGATFYNQKFKGKATFTVDTSSSTAYMKFNSLTFEDSAVYYCARLPIQFGNFYPMDYWGQGTSVTVSS.

[0371] The sequence of the light chain of the anti-MDR1 9F11 antibody used as described herein is as follows. DVLMTQTPLSLPVSLGDQASISCRSSQSIVHRTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK.

[0372] The sequence of the heavy chain of the anti-MDR1 9F11 antibody used as described herein is as follows. EVKLVESGGGLVKFGGSLKLSCAASGFTLSSYYMSWVRQSPEKRLELVAVINSNGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALYYCARPFYYSNSPFAYWGQGTLVTVSS.

[0373] The sequence of the heavy chain of the anti-MDR1 MM4.17 antibody used as described herein is as follows. QVQLQESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVATISSGGSYTYFPDSVKGRFTISRDNAKNTLYLQVSSLKSEDTAMYYCARPAEFRGYSWFAYWGQGTTVTVSS.

[0374] The sequence of the light chain of the anti-MDR1 M89 antibody used as described herein is as follows. EIVLTQSPATLSLSPGERATLSCRASQSVGGSYLAWYQQKPGQAPRLLIYGASRRATGIPARFSGSGSGTDFTLTISSLQPEDFASYFCQQTNTFPLTFGGGTKVEIK.

[0375] The sequence of the heavy chain of the anti-MDR1 M 89 antibody used as described herein is as follows. QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQDPGKGLMWVSSISTDGSATKYADSVKGRFTISRDNAKNTVSLQMNSLRAEDTAVYYCVGGFLGWWGQGTLVTVSS.

[0376] Results

[0377] [Biophysical Analysis Test of Purified mAb (GXII Apparatus, Reduced and Non-Reduced Proteins)] The purity and monomer content of the final protein preparation were measured by high-throughput analysis on a Caliper LabChip GXII using a Protein Express LabChip Kit (Perkin-Elmer) as described by the manufacturer. The chip was automatically primed on the instrument using a polymer solution containing 0.2% SDS and a fluorescent staining dye. The destaining channel was filled with a polymer solution without SDS and dye. Briefly, proteins under reducing and non-reducing conditions were prepared by mixing a small amount (2 - 5 μL) of sample with the Caliper sample buffer with or without DDT. The samples were denatured at 75 °C for 5 minutes, centrifuged at 2000 g for 3 minutes, and then electrophoresed. An electropherogram was generated by the LabChip GXII Touch software (Perkin Elmer).

[0378] [Detection of Specific Binding of CD47 and ABCB1] The binding specificities of monoclonal antibodies, Fabs, and bispecific IgG1 targeting CD47 were tested by ELISA (R&D systems) using human CD47-Fc fusion protein. Briefly, microtiter plates were coated with 50 μl of 0.5 μg / ml purified human CD47-Fc fusion protein in PBS and then blocked with 100 μl of 0.4% BSA in PBS. Dilutions of various antibody formats were added to each well in 1 / 3 serial dilutions and incubated at room temperature for 1 hour. A known 5F9 anti-CD47 antibody was used as a positive control, and human IgG1 was used as an isotype control. The plates were then washed three times with PBS / Tween and incubated with an HRP-conjugated donkey anti-human constant region-specific secondary reagent at room temperature for 1 hour. After washing, the plates were developed with an HRP substrate. The reaction was stopped with 2 M H2SO4, and the OD was measured at 520 nM.

[0379] The binding specificities of mAb, Fab, and bispecific IgG1 were tested by FACS using 293T cell lines that naturally express CD47, 293T naive cells that overexpress the human ABCB1 target, 293T naive cells in which ABCB1 was knocked down using a lentiviral RNA vector, CD47 knockout 293T cells, and CD47 knockout 293T cells that overexpress human ABCB1. Briefly, different cell lines were incubated on ice for 1 hour with various amounts of mAb or bispecific mAb or human IgG1 isotype control antibody. The cells were 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 an additional 1 hour. The samples were washed and analyzed using a BD FACS Canto (BD Biosciences).

[0380] This example shows the construction of a bispecific heterobivalent antibody molecule in which one arm binds to the efflux pump MDR1 / Pgp and the other arm binds to the cancer checkpoint protein CD47. When both targets are present on the cell surface simultaneously, this bispecific antibody binds to the cell with relatively high affinity / binding. In comparison, when either MDR1 / Pgp or CD47 is absent or substantially reduced, the binding of the bispecific antibody is significantly reduced or undetectable. (Figure 4).

[0381] The constructed bispecific antibody is shown in Figure 2 and contains one arm (arm A) that binds to and antagonizes the transporter protein (efflux pump Pgp), making the cell more sensitive to chemotherapeutic agents. At the same time, the other arm (arm B) binds to the "immune-Don't eat me" signal (CD47) on the cell surface, which, together with the IgG1 Fc, enables a stronger immune response against the cell.

[0382] Figures 3A - 3F provide FACS binding diagrams of various reagent cell lines used to test candidate molecules. Some of these reagent cell lines were engineered by knockdown, knockout, or overexpression of the target protein to allow for an accurate and clear understanding of the target involved, its presence or absence, and the cell's sensitivity to various anti-tumor drugs, as described in the Methods and Materials section. Specifically, Figure 3A shows binding to naive 293T cells, Figure 3B shows binding to 293T CD47-KO (Knock-out) cells, Figure 3C shows binding to 293T Pgp-KD (Knock-down) cells, Figure 3D shows binding to 293T Pgp-ox ("over-expressors"), Figure 3E shows binding to N6ADR (NALM6 Adriamycin resistant) cells, and Figure 3F shows binding to 293T CD47-KO+Pgp-ox cells.

[0383] In Figures 4A - 4B, the binding of the bispecific antibody is shown by FACS analysis on various cell lines. The first scan (left panels of Figures 4A and 4B) shows the binding of the antibody to cells co - expressing both targets (Pgp and CD47). The next two scans (central and right panels of Figures 4A and 4B) show the absence or significant decrease in binding when either Pgp (“KBP1”, right panel) or CD47 (“KT14”, central panel) of the cell line is knocked down (KD) or knocked out (KO). Figures 4A - 4B further show the binding at titrated antibody concentrations of 600 nM, 200 nM, 66 nM, 7.4 nM, and 2 nM, and also provide the signals measured from isotype and antibody - free controls. Each antibody concentration is shown in the left - hand panels of Figures 4A and 4B, and the signals measured from these different concentrations generally overlap in the central and right - hand panels of Figures 4A and 4B. Figures 4A and 4B show the results obtained from two different batches of antibodies.

[0384] Figures 5 and 6A - 6C show enhanced cell killing by the anti - tumor agent paclitaxel when cells are bound by the 15D3.aCD47:MRK16 bispecific antibody (highlighted by arrows). The data provided in Figure 5 show that the sensitivity of naive 293T cells and N6ADR cells to chemotherapy is enhanced by the 15D3.aCD47:MRK16 bispecific antibody compared to, for example, treatment with chemotherapy alone (“Chemo only”) or chemotherapy combined with other antibodies (15D3:MRK16 Fab; a15D3:MRK16 antibody; aCD47:MRK16 antibody; aCD47:MRK16 Fab; and 15D3:9F11 Fab).

[0385] Figure 6A provides a goodness-of-fit plot of the data provided in Figure 5, generated using GraphPad Prism software (GraphPad Software, Inc.). Figure...

Claims

**Claim 1** A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and CD47, 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, the first VH chain comprises an antigen-binding site for MDR1, the second VH chain comprises an antigen-binding site for CD47, and the second VH chain binds to CD47 when paired with one of the VL chains, the antigen-binding site of the first VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 9) comprising heavy chain CDRs 1-3 (HCDR 1-3) of the VH chain having, where X 2 is N, Q or S, the antigen-binding site of the second VH chain having the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 17) and comprising HCDR 1-3 of a VH chain having the same, the bispecific antibody molecule binds to cancer cells expressing both MDR1 and CD47, but exhibits reduced binding to non-cancer cells expressing MDR1 and / or CD47, the antigen-binding sites of the two VL chains having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 1) comprising light chain CDRs 1-3 (LCDR 1-3) of the VL chain having, where X 1 is N, Q or S, where the CDRs are CDRs according to the definitions of Kabat, Chothia, or MacCallum, A bispecific antibody molecule. **Claim 2** The bispecific antibody molecule according to claim 1, wherein the two VL chains are humanized and / or comprise an amino acid sequence that is at least 90% identical to the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 8); DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO: 3); or DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO: 4). **Claim 3** The bispecific antibody molecule according to claim 1, wherein the first VH chain is humanized and / or comprises an amino acid sequence that is at least 90% identical to the following amino acid sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 16); EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 11); or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 12). **Claim 4** The bispecific antibody molecule according to claim 1, wherein the second VH chain is humanized and / or comprises an amino acid sequence that is at least 90% identical to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 17), EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 21), EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 22), or QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 23). **Claim 5** A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and PD-L1, 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, the first VH chain comprises an antigen-binding site for MDR1, the second VH chain comprises an antigen-binding site for PD-L1, and the second VH chain binds to PD-L1 when paired with one of the VL chains, the antigen-binding site of the first VH chain being the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 9) comprising heavy chain CDRs 1 to 3 (HCDR 1 to 3) of a VH chain having, where X 2 is N, Q or S, the antigen-binding site of the second VH chain being the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 32) comprising HCDR 1-3 of a VH chain containing the same, the antigen-binding sites of the two VL chains being the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 1) comprising light chain CDRs 1 to 3 (LCDR 1 to 3) of the VL chain having 1 where X is N, Q or S, wherein the CDR is a CDR according to the definition of Kabat, Chothia, or MacCallum, a bispecific antibody molecule. **Claim 6** A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and EGFR, 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, the first VH chain comprises an antigen-binding site for MDR1, the second VH chain comprises an antigen-binding site for EGFR, and the second VH chain binds to EGFR when paired with one of the VL chains, the antigen-binding site of the first VH chain being the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 9) comprising heavy chain CDRs 1 to 3 (HCDR 1 to 3) of a VH chain having, wherein X 2 is N, Q or S, The antigen-binding site of the second VH chain is the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 36) comprising HCDR 1-3 of the VH chain containing the same, The antigen-binding sites of the two VL chains are the following sequences: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 1) comprising light chain CDRs 1 to 3 (LCDR 1 to 3) of the VL chain having, where X 1 is N, Q or S, wherein the CDR is a CDR according to the definition of Kabat, Chothia, or MacCallum, Bispecific antibody molecule. **Claim 7** The bispecific antibody molecule according to claim 1, wherein the antibody molecule comprises an Fc domain modified to reduce or eliminate the binding of the antibody molecule to one or more Fcγ receptors. **Claim 8** A pharmaceutical composition comprising the bispecific antibody molecule according to claim 1 for use in a method of treating a subject for cancer, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody molecule. **Claim 9** The pharmaceutical composition according to claim 8, wherein the method comprises administering the bispecific antibody molecule in combination with at least one additional active agent, and the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. **Claim 10** The pharmaceutical composition according to claim 9, wherein the at least one additional active agent is a chemotherapeutic agent. **Claim 11** The pharmaceutical composition according to claim 10, wherein the subject to be treated is a subject having cancer determined to be resistant to treatment with the chemotherapeutic agent. **Claim 12** The antibody molecule according to claim 1, wherein the first VH chain is fused to a first Fc domain and the second VH chain is fused to a second Fc domain. **Claim 13** The antibody molecule according to claim 1, and a pharmaceutically acceptable excipient comprising a pharmaceutical composition. **Claim 14** One or more nucleic acids comprising one or more sequences encoding the antibody molecule according to claim 1. **Claim 15** A mammalian cell genetically engineered with one or more nucleic acids according to claim 14. **Claim 16** The cell according to claim 15, wherein the cell is an immune cell. **Claim 17** The antibody molecule according to claim 1 or a nucleic acid encoding the same, and at least one additional active agent comprising a kit. **Claim 18** The pharmaceutical composition according to claim 8, wherein the cancer is drug-resistant or multi-drug resistant.

19. The pharmaceutical composition according to claim 10, wherein the chemotherapeutic agent is taxol, vinca alkaloid, or anthracycline.

20. The pharmaceutical composition according to claim 11, wherein the chemotherapeutic agent comprises paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide or nilotinib.

21. The antibody molecule according to claim 12, wherein the Fc domain comprises a modified CH3 domain that preferentially forms a heterodimer containing the first and second VH chains.

22. The antibody molecule according to claim 21, wherein the first and second Fc domains are human immunoglobulin G1 (IgG1) Fc domains.

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