Effulsion pump-cancer antigen multispecific antibody and related compositions, reagents, kits and methods

Abstract

A multispecific antibody targeting both a cell efflux pump and a cancer-related antigen is provided, as well as a pharmaceutical composition, nucleic acid, recombinant expression vector, cell, and kit comprising or encoding such a multispecific antibody. A method for treating a subject for cancer is also provided, comprising the step of administering the multispecific antibody targeting both a cell efflux pump and a cancer-related antigen to the subject. A method for producing the described multispecific antibody and a reagent associated therewith is also provided, comprising a genetically modified cell line useful for the target method and a method for producing such a genetically modified cell line.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] The present application claims the benefit of priority to U.S. Provisional Application No. 62 / 967,478 filed on January 29, 2020 and U.S. Provisional Application No. 62 / 828,044 filed on April 2, 2019, the full text of which is incorporated herein by reference. Background Technology

[0003] Introduction

[0004] Drug resistance, a widely known phenomenon that occurs when a disease develops resistance to pharmaceutical treatment, is a major and growing challenge in various fields of medicine, including oncology. While some mechanisms of drug resistance are disease-specific, others, such as drug efflux observed in microbial and human drug-resistant cancers, are evolutionarily conserved. Many types of cancer are initially sensitive to chemotherapy, but over time, they can develop resistance through these and other mechanisms, such as metabolic changes and DNA mutations, which promote drug inhibition, degradation, and enhanced efflux.

[0005] Effluent pumps (EPs) are proteins expressed by almost all living cells and have evolved to naturally efflux various compounds from the cell. Members of the ATP-binding cassette (ABC) transporter family are examples of EPs that enable drug efflux from the plasma membrane of healthy cells and are widely studied important regulators. Although the structure of transporters varies from protein to protein (for example, there are 49 known members of the ABC family in humans), they are all classified by the presence of two distinct domains: a highly conserved nucleotide-binding domain and a more variable transmembrane domain. Multidrug resistance protein 1, 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 increased in specific tissues (e.g., colon, liver, and kidney) when they became neoplastic, and increased expression in response to treatment with specific chemotherapy agents demonstrates that both intrinsic and extrinsic mechanisms of MDR1 overexpression are at work.

[0006] EP enables cells and tumors to develop resistance to chemotherapy drugs. This resistance is frequently associated with enhanced efflux of therapeutic molecules from resistant cells. When this chemotherapy resistance is applied to more than one chemotherapy drug, it is called multidrug resistance (MDR). Although various small molecule inhibitors targeting and inhibiting EP have been developed, none have been successful in human clinical settings for various reasons, including the tendency of these inhibitors to penetrate and affect all cells in the body, including healthy cells that utilize EP for the efflux of naturally occurring cytotoxins, regardless of the function of the cells or their efflux pumps.

[0007] Among cancer patients, when a population of metastatic cancer cells is largely eradicated and eliminated from the patient by the use of chemotherapy, it is not uncommon for a drug-resistant cancerous population of cells to emerge and spread without response to resumed treatment using the previous regimen. In most cases, different drugs with different mechanisms of action are applied until another new population of drug-resistant cells and / or tumors emerges.

[0008] An anti-MDR1 antibody that can be used as a multispecific antibody targeting both MDR1 and tumor-associated antigens (TAAs), as well as pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits comprising or encoding such a multispecific antibody, are provided. The multispecific antibody comprises a common variable light chain (VL) containing an antigen-binding site for MDR1, a first variable heavy chain (VH) containing an antigen-binding site for MDR1, and a second VH chain containing an antigen-binding site for TAA. A method for treating a subject for cancer is also provided, comprising the step of administering a multispecific antibody targeting both MDR1 and TAA to the subject. The treatment may involve the step of administering the multispecific antibody alone or the step of administering the multispecific antibody and a chemotherapy agent. A method for producing the described multispecific antibody and a reagent associated therewith is also provided, comprising a genetically modified cell line useful for the target method and a method for producing such a genetically modified cell line.

[0009] The bispecific antibody provided herein binds to cancer cells expressing both MDR1 and TAA, but exhibits reduced binding to non-cancer cells expressing MDR1 and / or TAA. In other words, the bispecific antibody provided herein binds with low affinity to (1) cells expressing TAA when MDR1 expression is low or absent and (2) cells expressing MDR1 when TAA expression is low or absent, and binds with high affinity to cancer cells expressing at least one or both of MDR1 and CD47 at relatively high levels, i.e., higher than normal cells.

[0010] In addition, an anti-MDR1 antibody having reduced affinity for MDR1 compared to the anti-MDR1 antibody 15D3 and an anti-CD47 antibody having reduced affinity for CD47 compared to the anti-CD47 antibody 5F9 are disclosed herein. These antibodies are used to treat cancer in subjects when co-administered and to increase the chemosensitivity of cancer cells to chemotherapy. Brief explanation of the drawing

[0011] FIG. 1A provides a schematic diagram of a specific example of the multispecific antibody of the present invention as described herein. FIG. 1B provides a schematic diagram showing the binding of a specific example of the multispecific antibody shown in FIG. 1A to a cell expressing one or both of the targets to which the multispecific antibody is targeted. FIG. 2 provides a schematic diagram of a bispecific monoclonal antibody described herein. Figures 3A-3F provide FACS analysis of the binding of labeled monoclonal antibodies to Pgp and CD47 (sometimes referred to as "KT14") on various cell lines, including naive and recombinant cell lines expressing the targets as shown. Figures 4A-4B provide FACS analysis of the binding of a bispecific antibody (HC-15D3:HC-5F9:LC-MRK16) to various reagent cell lines (e.g., 293T naive cells) that exhibit specific binding when both targets are co-expressed, and to reagent cell lines (e.g., 293 KT14-KO and KPB1-KD (Pgp is sometimes referred to as "KPB1")) that exhibit significantly reduced binding when only one of the targets is present. Figure 5 provides a chemosensitivity assay showing the sensitivity of Naive 293T and N6ADR cell lines to paclitaxel in the presence of various monovalent, Fab, and bispecific antibody molecules described herein. Cells (Naive 293T cells (left panel) and N6ADR cells (right panel)) exhibited enhanced sensitivity in the presence of the 15D3.aCD47:MRK16 bispecific antibody compared to treatment with chemotherapy alone ("Chemo alone") or chemotherapy combined with other antibodies (15D3:MRK16 Fab; a15D3:MRK16 antibody; aCD47:MRK16 antibody; aCD47:MRK16 Fab; and 15D3:9F11 Fab). Figure 5 demonstrates that bispecific antibodies improve the chemosensitivity of Naive 293T cells and Adriamycin-resistant N6ADR cells, even with reduced affinity of the bispecific antibodies to individual targets. Figures 6A-6C provide a re-representation of the data provided in Figure 5 and include a side-by-side fitted curve of Naive 293T cell and N6ADR cell data (Figure 6A) and a side-by-side drawing of raw (left) and fitted (right) data for Naive 293T cells (Figure 6B) and N6ADR cells (Figure 6C). Figure 7 demonstrates the drug sensitization effect of the 15D3.aCD47:MRK16 antibody when used in combination with vincristine. Figure 8 provides a table showing the binding and target cell killing of tested antibody light chain and heavy chain combinations as described in the Examples section. Figure 9 shows the effect of the indicated antibody on the tumor volume of MES-SA-DX5 (uterine sarcoma) without chemotherapy. Figure 10 shows the effect of paclitaxel on inhibiting tumor growth in NALM6 cells. Figure 11 shows the effect of BisP1.1 on inhibiting tumor growth in paclitaxel-resistant NALM6ADR cells. Figure 12 shows the effect of valspodar on inhibiting tumor growth in paclitaxel-resistant NALM6ADR cells. Figure 13 shows the effect of BisP1.1 on the body weight of mice transplanted with paclitaxel-resistant NALM6ADR cells. Figure 14 shows the effect of valspodar on the body weight of mice transplanted with paclitaxel-resistant NALM6ADR cells. Figure 15 shows the effect of KBisP1.1 on the survival of mice transplanted with paclitaxel-resistant NALM6ADR cells. Figure 16 shows the expression of CD47 and ABCB1 on the surface of A2780ADR cells. Figure 17 shows the expression of CD47 and ABCB1 on the surface of HeyT30 cells. Figure 18 shows the reduction in A2780ADR tumor volume in vivo after administration of KBisP1.1 with or without paclitaxel. Figure 19 shows the reduction in HeyT30 tumor volume in vivo after administration of KBisP1.1 with or without paclitaxel. Figure 20 shows that KbisP1.1 is more effective than the 15D3 antibody against N6ADR cell tumors in vivo. Figure 21 shows that chemosensitization of KbisP1.1 to paclitaxel in multidrug-resistant N6ADR tumors is dose-dependent in vivo. Figures 22A-22B show that the KBisP1.1 antibody does not show significant binding to human red blood cells (RBCs) (Figure 22A) and cynomolgus RBCs (Figure 22B) in contrast to the anti-CD47 5F9 antibody, and that the anti-CD47 antibody ("KT14_MRK16") containing 5F9 HC and MRK16 LC does not significantly bind to RBCs in contrast to the anti-CD47 5F9 antibody ("KT14"). Figure 23 shows the alignment of the humanized 15D3 VH chain sequence. Figure 24 shows the alignment of the humanized MRKK16 chain sequence. Figure 25 shows that a humanized muted Fc bispecific antibody chemosensitizes multidrug-resistant MES-SA-DX5 tumors. Figure 26 shows that the humanized KBisP1.1 antibody chemosensitizes multidrug-resistant A2780ADR cell-generated tumors in mice. Figure 27A shows that the N297A-substituted KBisP1.1 bispecific antibody chemosensitizes multidrug-resistant MES-SA / Dx5 cell tumors in mice to paclitaxel. Figure 27B shows that the KBisP1.1 N297A antibody (labeled "N297A") sensitizes multidrug-resistant A2780ADR cells to paclitaxel in vivo. Figure 28 shows that the KBisP1.1 antibody sensitizes multidrug-resistant A2780ADR cells to paclitaxel in vivo. Figure 29 shows that the KBisP1.1 antibody sensitizes multidrug-resistant MES-SA / Dx5 cells to paclitaxel in vivo. Figure 30 shows that the KBisP1.1 bispecific antibody sensitized paclitaxel-resistant CTG-2616 patient-derived breast tumors in vivo and inhibited tumor growth. Figure 31 provides an overview of the binding of the labeled antibodies to CD47 measured by ELISA, MDR1 measured by FACS, PD-L1 measured by ELISA, and EGFR measured by ELISA or FACS. Figure 32A shows the MFI for the listed antibodies measured using 293T cells overexpressing MDR1. Figure 32B shows the MFI for the listed antibodies measured using 293T cells overexpressing EGFR. Figure 32C shows the MFI for the listed antibodies measured using 293T cells overexpressing EGFR against a subset of antibodies shown in Figure 32B. Specific details for implementing the invention

[0012] definition

[0013] The terms “antibody” and “immunoglobulin” include any isotype of antibody or immunoglobulin, fragments of an antibody that maintain a specific binding to an antigen, and, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, such as antibodies containing only the heavy chain (e.g., VHH camelid antibody), bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies may be detectably labeled, for example, with radioisotopes, enzymes that produce detectable products, fluorescent proteins, etc. Antibodies may be further conjugated to other moieties, such as members of specific binding pairs, for example, biotin (members of biotin-avidin specific binding pairs), etc. Antibodies may also be bound to solid supports, including, but not limited to, polystyrene plates or beads, etc. Additionally, the term includes Fab', Fv, F(ab')2, and / or other antibody fragments that maintain specific binding to antigens, and monoclonal antibodies. Antibodies may be monovalent or divalent. The antibodies used herein may be used to analyze the expression of target antigen(s) on a cell surface, for example, in a patient's cell sample or tissue sample.

[0014] “Antibody fragments” are parts of an intact antibody, for example, containing 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, such as antibodies containing only the heavy chain (e.g., VHH camelid antibody); and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments called “Fab” fragments, each having a single antigen-binding site and a residual “Fc” fragment, a designation reflecting the ability to easily crystallize. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites and is capable of cross-linking antigens.

[0015] "Fv" is a minimal antibody fragment containing a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain that are tightly non-covalently associated. The three CDRs of each variable domain interact to form V H -V L The delimitation of antigen-binding sites on the surface of the dimer is achieved in this configuration. Overall, six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of the Fv containing only three antigen-specific CDRs) can form an antigen-binding site with the ability to recognize and bind to the antigen, even at a lower affinity than the entire binding site containing three CDRs of each variable domain.

[0016] The "Fab" fragment also contains a constant domain of the light chain and a 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, which contains one or more cystes in the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domain contain free thiol groups. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments having hinge cystes between them. Other chemical couplings of antibody fragments are also known.

[0017] The "light chain" of an antibody (immunoglobulin) of any vertebrate species can be assigned to one of two distinct types called kappa and lambda based on the amino acid sequence of the constant domain. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, many of which can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0018] "Single-stranded Fv", "sFv", or "scFv" antibody fragments are the antibody's V H and V L It contains domains, and these domains exist on a single polypeptide chain. In some embodiments, the Fv polypeptide is V H and V L It further includes polypeptide linkers between domains, enabling the sFV to form a structure favorable for antigen binding. For the examination of the sFV, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) ) reference.

[0019] The term "diabadi" refers to a small antibody fragment having two antigen-binding sites, and this fragment has a light chain variable domain (V) within the same polypeptide chain. L Heavy chain variable domain (V) connected to ) H ) (V H -V L ...includes ). By using a linker that is too fine to allow pairing between two domains on the same chain, the domain is forced to pair with a complementary domain on another chain to create two antigen-binding sites. Diabodies are, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) It is described more sufficiently in [link].

[0020] As used herein, the term “affinity” represents the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (Kd). Affinity may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 1000 times greater than the affinity of the antibody to an unrelated amino acid sequence. The affinity of the antibody for the target protein may be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM) or greater. As used herein, the term “binding strength” indicates the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactive” and “preferentially bind” are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0021] The term "binding" refers to the direct association between two molecules resulting from covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions, including interactions such as, for example, salt bridges and water bridges. MDR1-specific antibodies specifically bind to epitopes within the MDR1 polypeptide. Non-specific binding is approximately 10 -7 Affinity less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It will represent the combination with affinities such as M, etc.

[0022] As used herein, the term “CDR” or “complementary determining region” is intended to mean non-adjacent antigenic combination 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., US Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definition includes an overlap or subset of amino acid residues when compared to one another. Nevertheless, the application of the definition to denote the CDR of an antibody or a transplanted antibody or a variant thereof is intended to be within the scope of the term as defined and used herein. Amino acid residues containing the CDR as defined by each of the cited references above are presented in Table 1 below for comparison.

[0023] CDR definition Kabat 1 Chothia 2 MacCallum 3 IN H CDR1 31-35 26-32 30-35 In H CDR2 50-65 53-55 47-58 In H CDR3 95-102 96-101 93-101 IN L CDR1 24-34 26-32 30-36 In L CDR2 50-56 50-52 46-55 In L CDR3 89-97 91-96 89-96

[0024] 1 Residual numbering is Kabat et al., supra It follows the naming convention.

[0025] 2 Residual numbering is Chothia et al., supra It follows the naming convention.

[0026] 3 Residual numbering is MacCallum et al., supra It follows the naming convention.

[0027] As used herein, the term “framework” is intended to mean all amino acid residues outside the CDR region within the antibody’s variable region when used with respect to the antibody variable region. The variable region framework is generally a discontinuous amino acid sequence of about 100–120 amino acids in length, but is intended to refer only to said amino acids outside the CDR. As used herein, the term “framework region” is intended to mean each domain of the framework separated by the CDR. The VH chain may contain three CDRs and four FRs arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, the VL chain may contain three CDRs and four FRs arranged in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0028] As used herein, the term antibody comprises a tetramer of two heavy chains and two light chains, the heavy chains and light chains being connected to each other, for example, by disulfide bonds. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains comprise binding regions that interact with antigens. The constant region of the antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system and first components of the complement system. The term “antibody” includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM, and their subtypes. In some embodiments, the target antibody is an IgG isotype, for example, IgG1.

[0029] 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 many immunoglobulin variable region genes. The 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. The 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 aforementioned constant region genes at the C-terminus, e.g., gamma (encoding about 330 amino acids). In some embodiments, the target antibody comprises a whole immunoglobulin comprising a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0030] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody capable of specifically binding to an antigen. Examples of binding fragments include (i) a Fab fragment (a monovalent fragment containing VL, VH, CL, and CH1 domains, e.g., consisting of these); (ii) an F(ab')2 fragment (a divalent fragment containing two Fab fragments connected by a disulfide bridge at a hinge region); (iii) an Fd fragment (containing VH and CH1 domains, e.g., consisting of these); (iv) an Fv fragment (containing VH and VL domains of a single arm of the antibody, e.g., consisting of these); (v) a dAb fragment (containing a VH domain, e.g., consisting of this); (vi) an isolated CDR; (vii) single-stranded Fv (scFv) (comprising the VH and VL domains of a single arm of an antibody combined by a synthetic linker using recombinant means so that the VH and VL domains pair to form a monovalent molecule, e.g., composed of these); (viii) diabody (comprising two scFvs in which the VH and VL domains do not pair to form a monovalent molecule, e.g., composed of these; wherein the VH of each of the scFv pairs with the VL domain of the other scFv to form a divalent molecule).

[0031] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0032] "Human antibody" is one that has an amino acid sequence corresponding to an antibody produced by a human or human cell, or derived from a non-human source utilizing the 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.

[0033] The "Human Common Framework" is a framework (FR) representing the amino acid residues that most commonly occur when selecting human immunoglobulin variable light chain (VL) or variable heavy chain (VH) framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from subgroups of variable domain sequences. Generally, the subgroups of the sequences are those found in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I, as described above by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III, as described above by Kabat et al.

[0034] "Humanized" antibodies represent chimeric antibodies comprising amino acid residues of non-human CDRs and amino acid residues of the human framework (FR). At least a portion of the humanized antibody constant region is derived from a human antibody. In a preferred embodiment of the antibody molecule disclosed herein, the constant region is derived from a human IgG antibody, e.g., human IgG1. In a preferred embodiment, the antibody molecule disclosed herein comprises a heavy chain comprising a variable heavy chain region provided herein and a human IgG1 constant region having the amino acid sequence presented in UniProt: P01857-1, Version 1. In a preferred embodiment, the antibody molecule disclosed herein comprises a light chain comprising a variable light chain region provided herein and a human light chain constant region. In a preferred embodiment, the human light chain constant region is a human kappa light chain constant region. In certain embodiments, the human IgG1 heavy chain constant region present in the target antibody may include mutations, e.g., substitutions, to modulate Fc function. For example, LALAPG effector function mutations (L234A, L235A, and P329G) or the N297A mutation may be introduced to reduce antibody-dependent cytotoxicity (ADCC). The numbering of substitutions is based on the EU numbering system. The "EU numbering system" or "EU index" is generally used to denote residues in the immunoglobulin heavy chain constant region (e.g., 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 IgG 1 EU antibody.

[0035] The "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.

[0036] The term "epitope" refers to a region of an antigen recognized by the immune system, for example, antibodies, B cells, or T cells. For example, an epitope is a specific region of an antigen to which an antibody binds.

[0037] “Isolated” antibodies are identified and separated from and / or recovered from components of the natural environment. Contaminating components of the natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, antibodies will be purified to a homogeneous degree by (1) exceeding 90%, 95%, or 98% by weight of the antibody as determined by the Lowry method, e.g., exceeding 99% by weight, (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary cup array determining device, or (3) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue or silver staining under reducing or non-reducing conditions. The isolated antibody contains the antibody in situ within the recombinant cell because at least one component of the antibody's natural environment will be absent. In some cases, the isolated antibody will be prepared by at least one purification step.

[0038] The term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes apoptosis or destruction, as used herein. "Chemotherapy agents," also called "antineoplastic agents," may be cytotoxic agents used to treat cancer or other diseases or disorders.

[0039] As used herein, the terms “treatment,” “treating,” etc., indicate obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic with respect to completely or partially preventing a disease or its symptoms and / or therapeutic with respect to partial or complete cure of the disease and / or adverse effects attributable to the disease. “Treatment,” as used herein, includes any treatment of a disease in mammals, including humans, and includes (a) prevention of the occurrence of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with the disease; (b) suppression of the disease, i.e., cessation of its development; and (c) alleviation of the disease, i.e., inducing regression of the disease.

[0040] The terms “individual,” “subject,” “host,” and “patient,” as used interchangeably herein, refer to mammals including, but not limited to, rats (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc.

[0041] "Therapeutic effective dose" or "effective dose" refers to an amount sufficient for such treatment to have an effect on the disease when administered to mammals and other subjects to treat the disease. The "therapeutic effective dose" will vary depending on the antibody, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0042] As used herein, the term "intractable" refers to a disease or pathological condition that does not respond to treatment. With respect to cancer, "intractable cancer" refers to cancer that does not respond to treatment, as used herein. Intractable cancer may be resistant at the start of treatment or may become resistant during treatment. Intractable cancer may also be referred to as resistant cancer.

[0043] "Biological samples" include various types of samples obtained from an individual and may be used in diagnostic or monitoring analyses. The definition includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures, or cells and their offspring derived therefrom. The definition also includes samples manipulated by any method after their procurement, such as treatment with reagents, solubilization, or enrichment with specific components, such as polynucleotides. The term "biological samples" includes clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0044] The percentage identity between sequence pairs 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. The identity score counts only perfect matches and does not consider the degree of similarity between amino acids. Only internal gaps are included in the length, while gaps at the ends of the sequences are excluded. Percent Identity = (Matches x 100) / Length of Aligned Region (including gaps).

[0045] The phrase “conservative amino acid substitution” refers to the substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Conservative amino acid substitution can preserve the activity of a protein by replacing amino acid(s) of the protein with amino acids having side chains of similar acidity, basicity, charge, polarity, or size.

[0046] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[0047] The terms “expression vector” or “expression construct” refer to a vector suitable for transformation of a host cell and contain nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions operably linked thereto (with the host cell). The expression construct may include, but is not limited to, sequences that influence or control transcription and translation, and, where introns are present, influence the RNA splicing of coding regions operably linked thereto.

[0048] The term “stimulation” refers to a primary response induced by the binding of a stimulating molecule (e.g., TCR / CD3 complex or CAR) to a homologous ligand (or tumor antigen in the case of CAR), which mediates signal transduction events such as signal transduction via the TCR / CD3 complex or signal transduction via the appropriate NK receptor or signal transduction domain of the CAR, but is not limited to. Stimulation can mediate altered expression of specific molecules.

[0049] The term “stimulatory molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides cytoplasmic signaling sequence(s) that regulate the activation of immune cells by a stimulatory method for at least some modalities of immune cell signaling pathways. In one modality, the signal is a primary signal initiated by the binding of a TCR / CD3 complex with an MHC molecule loaded with, for example, a peptide, and leads to the mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence acting by a stimulatory method (also called the “primary signaling domain”) may contain a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing cytoplasmic signaling sequences specifically used in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0050] The term “co-stimulatory molecule” refers to a co-binding partner on a T cell that specifically binds to a co-stimulatory ligand and mediates a co-stimulatory response by the T cell, e.g., proliferation, but not limited to. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that contributes to an efficient immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0051] The term "self" refers to any substance derived from the same entity that is subsequently reintroduced as an entity.

[0052] "Intracellular signaling domain" refers to the intracellular portion of a molecule as the term is used herein. The intracellular signaling domain generates signals that promote immune effector functions in CAR-containing cells, e.g., CAR-T cells. For example, in CAR-T cells, examples of immune effector functions include cytolytic activity and helper activity, and include the secretion of cytokines.

[0053] "Immune effector cells" refers to cells involved in the promotion of an immune response, e.g., an immune effector response, as the term is used herein. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytic cells.

[0054] Guidelines for substitution, insertion, or deletion may be based on the alignment of amino acid sequences of proteins from common sequences based on different species or multiple proteins having the same or similar functions.

[0055] details

[0056] A multispecific antibody targeting both MDR1 and tumor-associated antigens (TAAs) is provided, as well as pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and anti-MDR1 antibodies that may be used as kits containing or encoding such a multispecific antibody. The multispecific antibody comprises a common variable light chain (VL) containing an antigen-binding site for MDR1, a first variable heavy chain (VH) containing an antigen-binding site for MDR1, and a second VH chain containing an antigen-binding site for TAA. A method for treating a subject for cancer is also provided, comprising the step of administering the multispecific antibody targeting both MDR1 and TAA to the subject. The treatment step may involve administering the multispecific antibody alone or administering the multispecific antibody and a chemotherapy agent. A method for producing the described multispecific antibody and related reagents are also provided, including a genetically modified cell line useful in the target method and a method for producing such a genetically modified cell line.

[0057] The bispecific antibody provided herein binds to cancer cells expressing both MDR1 and TAA, while exhibiting reduced binding to non-cancer cells expressing MDR1 and / or TAA. In other words, the bispecific antibody provided herein binds with low affinity to (1) cells expressing TAA when MDR1 expression is low or absent and (2) cells expressing MDR1 when TAA expression is low or absent, and binds with high affinity to cancer cells expressing at least one or both of MDR1 and CD47 at relatively high levels, i.e., higher than normal cells.

[0058] Anti-MDR1 antibodies exhibiting reduced affinity for MDR1 compared to anti-MDR1 antibody 15D3 and anti-CD47 antibodies exhibiting reduced affinity for CD47 compared to anti-CD47 antibody 5F9 are also disclosed herein. These antibodies are used in the treatment of cancer in subjects to increase the chemosensitivity of cancer cells to chemotherapy when co-administered.

[0059] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described herein and, of course, may vary. It should also be understood that the terms used herein are intended only for the purpose of describing specific embodiments, and that there is no intention to limit the scope of the invention, as the scope of the invention is limited only by the appended claims.

[0060] Where a range of values ​​is provided, between the upper and lower limits of said range and any other mentioned value or between any intermediate value within said range, each intermediate value is understood to be included within the present invention up to one-tenth of the unit of the lower limit, unless otherwise clearly indicated by the context. The upper and lower limits of these smaller ranges may be included in an equally smaller range and are also included within the present invention subject to any specifically excluded limits from said range. Where said range includes one or both of the limits, the range excluding one or both of said included limits is also included in the present invention.

[0061] A specific range having a number preceded by the term “approx.” is provided. The term “approx.” is used herein to provide literal support for the exact number preceded by the term, as well as for a number near or approximate to the number preceded by the term. In determining whether a number is a number near or approximate to a specifically listed number, the near or approximate unlisted number may be a number that provides a substantial equivalent to the specifically listed number in the context in which it is provided.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains. Any method and material similar or equivalent to those described herein may also be used in the practice or testing of the invention, but representative examples of methods and materials are described hereafter.

[0063] All publications and patents cited herein are incorporated by reference as they appear to be specifically and individually incorporated herein, and are incorporated by reference herein to disclose and describe methods and / or materials related to the cited publications. Any citation of any publication is to a disclosure prior to the filing date and should not be construed as an acknowledgment that the present invention is not qualified to precede such publication. Additionally, the provided disclosure dates may differ from the actual disclosure dates and need to be independently verified.

[0064] As used herein and in the appended claims, the singular forms “one (a),” “one (an),” and “the” include multiple referents unless otherwise clearly indicated in the context. Additionally, claims may be drafted to exclude any optional elements. As such, these statements are intended to serve as a basis for the use of exclusive terms such as “only,” “only,” etc., or “negative” limitations with respect to the listing of claim elements.

[0065] As will be apparent to those skilled in the art when reading the present invention, the individual embodiments described and illustrated herein have distinct components and features that can be easily distinguished or combined with features of various other embodiments without departing from the scope or spirit of the invention. Any listed method may be performed in the order of listed events or in any other logically possible order.

[0066] Methods and compositions have been or will be described for grammatical fluidity with functional descriptions, but it should be clearly understood that claims must not be interpreted as being limited in any way by the composition of "means" or "steps" limitation unless explicitly defined under 35 USC §112(f), but must be in accordance with the meaning of the definition provided by the claim and the full scope of equivalents under the judicial principle of equivalents, and where explicitly defined under 35 USC §112(f), must be in accordance with the full statutory equivalents under 35 USC §112(f).

[0067] antibodies

[0068] bispecific antibody

[0069] The present invention provides a bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and tumor-associated antigen (TAA), wherein the antibody molecule comprises two identical variable light chains (VL), a first variable heavy chain (VH), and a second VH chain, wherein the 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 expressing both MDR1 and TAA but exhibits reduced binding to non-cancer cells expressing MDR1 and / or TAA. In other words, the bispecific antibody provided herein binds with low affinity to (1) cells expressing TAA when MDR1 expression is low or absent and (2) cells expressing MDR1 when TAA expression is low or absent, and binds with high affinity to cancer cells expressing at least one or both of MDR1 and CD47 at a relatively high level, that is, at a higher level than normal cells.

[0070] A relatively high level indicates expression that is at least 1.5 times (e.g., at least 2X, at least 3X, at least 4X, at least 5X, or more) the expression level in normal cells of the same type as cancer cells. Reduced affinity indicates 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 cancer cells. Reduced affinity also includes a lack of detectable binding.

[0071] The term “antibody molecule” comprises the antibody defined herein and its antigen-binding fragment. In certain embodiments, the antibody molecule comprises two variable light chains (VL) and two variable heavy chains (VH). In certain embodiments, the antibody molecule also comprises heavy chain and light chain constant regions. The heavy chain and light chain constant regions may be derived from human antibodies, for example, human IgG1 antibodies. The human IgG1 heavy chain (HC) constant region may be modified to include mutations that reduce antibody-dependent cytotoxicity (ADCC). In addition, or alternatively, the two VH chains may each be conjugated to different human IgG1 HC constant regions having substitutions that favor the formation of dimers between different human IgG1 HC constant regions. These HC regions are described in more detail herein. In a specific embodiment, where the antibody molecule is a bispecific antibody molecule, one of the human IgG1 HC constant regions may include a substitution for introducing one or more amino acids having a positively charged side chain, and the other human IgG1 HC constant region may include a substitution for introducing one or more amino acids having a negatively charged side chain that favors the formation of a dimer between two different HCs.

[0072] In a specific embodiment, the antigen-binding sites of the two VL chains comprise a light chain CDR (LCDR) of the VL chain having the following sequence:

[0073] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (here X 1 is N, Q, or S).

[0074] In a specific embodiment, the two VL chains comprise LCDR 1-3 of the VL chains having the following sequences:

[0075] (i) DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK;

[0076] (ii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; or

[0077] (iii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK.

[0078] In some embodiments, the antibody described herein comprises (i) two VL chains comprising an LCDR of a VL chain having the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; and a first VH chain comprising an HCDR having the following sequence. VH chain: does not contain EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS

[0079] In certain embodiments, the CDRs of the VL and VH light chains can be defined based on the Kabat nomenclature.

[0080] In a specific embodiment, i) LCDR1 is the following sequence: RSSQSIVHSTGX 1(ii) containing TYLE; (ii) LCDR2 contains the following sequence: KISNRFS; and (iii) LCDR3 contains the following sequence: FQASHFPRT; where X 1 is N, Q, or S. These LCDRs are based on the Kabat nomenclature.

[0081] In a specific embodiment, two VL chains are humanized. In a specific embodiment, two VL chains are humanized to include a framework region from a human antibody.

[0082] In a specific embodiment, the two VL chains contain a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or a 100% identical to the following sequence:

[0083] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK;

[0084] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; or

[0085] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK.

[0086] In a specific embodiment, the bispecific antibody molecule comprises two VL chains, each containing the following sequences:

[0087] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK.

[0088] In certain embodiments, the bispecific antibody comprises a light chain comprising the VL chain and a light chain constant region described herein. The light chain constant region may be a human immunoglobulin kappa chain constant region having the amino acid sequence presented in UniProtKB / Swiss-Prot: P01834.2.

[0089] In a specific embodiment, the bispecific antibody molecule comprises a first VH chain, and the VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (X 2 is N, Q, or S).

[0090] In a specific embodiment, the first VH chain comprises HCDR 1-3 of the VH chain having the following sequences:

[0091] (i) EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or

[0092] (ii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or

[0093] (iii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0094] In certain embodiments, HCDR 1-3 of the VH chain are defined based on Kabat nomenclature.

[0095] In a specific embodiment, the first VH chain comprises (i) HCDR1: RYTMS containing the following sequence; (ii) HCDR2: TISSGGGX containing the following sequence 2 TYYPDSVKG; and (iii) comprising HCDR3: YGAGDAWFAY containing the following sequence; where X 2 is N, Q, or S. These HCDRs are based on the Kabat nomenclature.

[0096] In a specific embodiment, the first and / or second VH chain is humanized. In a specific embodiment, the VH chain is humanized to include a framework region from a human antibody.

[0097] In a specific embodiment, the first VH chain comprises the following sequence:

[0098] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS;

[0099] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; or

[0100] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0101] In a specific embodiment, the first VH chain comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the following sequence:

[0102] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS;

[0103] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; or

[0104] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0105] In a specific embodiment, the bispecific antibody comprises a first heavy chain comprising the first VH chain and the human IgG1 heavy chain constant region described herein.

[0106] In a specific embodiment, the second VH chain of the bispecific antibody is derived from a monospecific antibody molecule that binds to TAA, and the affinity of the bispecific antibody molecule for TAA is at least 2 times (e.g., at least 3 times, at least 4 times, at least 5 times) lower than the affinity of the monospecific antibody molecule for TAA from which the VH chain is derived. The affinities of the bispecific antibody and the monospecific antibody are measured using the same assay. Any method suitable for measuring antibody affinity may be used. In a specific embodiment, affinity may be measured by calculating the equilibrium constant for the reversible binding of the antibody to the antigen and is expressed as a dissociation constant (Kd). In a specific embodiment, Kd may be measured by ELISA.

[0107] In a specific embodiment, the second VH chain of the bispecific antibody is derived from a monospecific antibody molecule that binds to TAA, and the half-maximum effective concentration (EC50) of the bispecific antibody molecule for TAA is at least 2 times (e.g., at least 3 times, at least 4 times, at least 5 times) higher than the EC50 of the monospecific antibody molecule for TAA from which the VH chain is derived. The EC50s of the bispecific antibody and the monospecific antibody are measured using the same assay. Any method suitable for measuring the EC50 of the antibody may be used. The concentration that provides a half-maximum response (e.g., half of the maximum fluorescence intensity) is measured as the EC50.

[0108] The EC50 of the test antibody can be determined by flow cytometry or ELISA. For example, flow cytometry may involve contacting cells expressing an antigen (e.g., human wild-type MDR1 or mutant MDR1) with an antibody in serially diluted flow cytometry buffer and incubating them at room temperature or 4°C for a period sufficient for the antibody to bind to the cells (e.g., 10 min to 1 hr). After incubation, the cells may be optionally washed to remove non-specifically bound antibodies and / or the cells may be contacted with a fluorescently labeled secondary antibody that specifically binds to the test antibody. After incubation, the fluorescently labeled secondary antibody may be removed and the cells may be washed. The washed cells are sorted by flow cytometry, and the number of cells bound to the fluorescently labeled secondary antibody may be counted. The concentration that provides a half-maximum response (e.g., half of the maximum fluorescence intensity) is measured as the EC50. As a variation of flow cytometry analysis, the cells may be 293T cells that overexpress MDR1.

[0109] TAA may be any antigen known to be overexpressed in cancer cells. For example, TAA may be an antigen that is expressed in cancer cells but not at detectable levels in normal cells, and normal cells and cancer cells are of the same cell type, e.g., epithelial cells. For example, TAA may be a neoantigen, which is a class of tumor antigens arising from tumor-specific mutation(s) that alter the amino acid sequence of a protein encoded by it compared to the amino acid sequence of an unmutated protein. In another embodiment, TAA is an antigen that is expressed in normal cells but at higher levels in cancer cells. In a specific embodiment, TAA may be CD47.

[0110] Anti-MDR1 and anti-CD47 bispecific antibodies

[0111] In a specific embodiment, the bispecific antibody molecule binds to CD47 and the second VH chain comprises an HCDR of the VH chain comprising the following amino acid sequence:

[0112] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0113] In a specific embodiment, the second VH chain comprises HCDR1: NYNMH containing the following sequence, HCDR2: TIYPGNDDTSYNQKFKD containing the following sequence, and HCDR3: GGYRAMDY containing the following sequence. HCDR1-3 are defined based on the Kabat nomenclature.

[0114] In a specific embodiment, the second VH chain comprises a sequence that is at least 80% (e.g., at least 85%, at least 90%, or at least 95%) identical to the following amino acid sequence:

[0115] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0116] In a specific embodiment, the second VH chain comprises the following amino acid sequence:

[0117] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0118] In a specific embodiment, a bispecific antibody binding to MDR1 and CD47 may comprise a humanized VH chain comprising an antigen-binding site for CD47 having the following sequence:

[0119] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS;

[0120] EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS; or

[0121] QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS.

[0122] Additional embodiments of the bispecific antibody are described elsewhere in this application and include a humanized version of the sequence variation disclosed herein and / or a substitution in the Fc region to promote the formation of a heterodimer between the first VH chain and the second VH chain.

[0123] Anti-MDR1 and anti-PD-L1 bispecific antibodies

[0124] In certain embodiments, TAA may be scheduled death-ligand 1 (PD-L1). PD-L1 is also known as differentiation 274 cluster (CD274) or B7 homolog 1 (B7-H1). In certain embodiments, a bispecific antibody molecule binding 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 comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0125] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS.

[0126] HCDR 1-3, defined according to the Kabat nomenclature, are as follows:

[0127] HCDR1: DSWIH

[0128] HCDR2: WISPYGGSTYYADSVKG

[0129] HCDR3: RHWPGGFDY.

[0130] The second VH chain of a bispecific antibody binding to MDR1 and PD-L1 may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the following amino acid sequence:

[0131] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS.

[0132] The second VH chain of a bispecific antibody binding to MDR1 and PD-L1 may be present in a heavy chain having an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the following amino acid sequence:

[0133] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0134] As described elsewhere in this invention, at least one, two, or all of the VL chain, the first VH chain, and the second VH may be humanized. In addition, the Fc region of the VH chain may include substitutions to increase heterodimerization between the first VH chain and the second VH chain. In a specific embodiment, the first heavy chain may include humanized and charged pair substitutions K392D and K409D, and the second heavy chain may include charged pair substitutions E356K and D399K.

[0135] Anti-MDR1 and anti-EGFR bispecific antibodies

[0136] In certain embodiments, the TAA may be the epidermal growth factor receptor (EGFR). HCDR1-3 for the second VH chain containing an antigen-binding site for EGFR may be derived from the VH chain of the anti-EGFR antibody necitumumab or cetuximab. The heavy chain of necitumumab has the following sequence:

[0137] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0138] 세툭시맙의 중쇄는 다음 서열을 갖는다:

[0139] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0140] In the first embodiment, the anti-MDR1 anti-EGFR bispecific antibody comprises a VL and a first VH chain as described in the previous section, and the second VH chain may comprise an HCDR from the VH region of necitumumab. The HCDR defined according to Kabat nomenclature may have the following sequence:

[0141] HCDR1: SGDYYWS

[0142] HCDR2: YIYYSGSTDYNPSLKS

[0143] HCDR3: VSIFGVGTFDY.

[0144] In a specific embodiment, the second VH chain may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the following amino acid sequence:

[0145] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS.

[0146] The second VH chain of a bispecific antibody binding to MDR1 and EGFR may be present in a heavy chain having an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the following amino acid sequence:

[0147] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQ GTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0148] In a second embodiment, the anti-MDR1 anti-EGFR bispecific antibody comprises a VL and a first VH chain as described in the previous section, and the second VH chain may comprise an HCDR from the VH region of cetuximab. The HCDR defined according to Kabat nomenclature may have the following sequence:

[0149] HCDR1: NYGVH

[0150] HCDR2: VIWSGGNTDYNTPFTS

[0151] HCDR3: ALTYYDYEFAY

[0152] In a specific embodiment, the second VH chain may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the following amino acid sequence:

[0153] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA.

[0154] The second VH chain of a bispecific antibody binding to MDR1 and EGFR may be present in a heavy chain having an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the following amino acid sequence:

[0155] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0156] The anti-MDR1 anti-EGFR bispecific antibody described above contains the same combination of VH and VL chains as the anti-MDR1 anti-CD47 bispecific antibody, and the anti-MDR1 anti-PD-L1 bispecific antibody described above may be called the 15D3 HC :: MRK16 LC :: Necitumumab HC and 15D3 HC :: MRK16 LC :: Cetuximab HC antibodies.

[0157] In addition, bispecific antibody molecules comprising 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 are provided herein, but are not based on MRK16 LC and / or 15D3 HC as described above.

[0158] In certain embodiments, the anti-MDR1 anti-EGFR bispecific antibody may 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.

[0159] The HCDR and LCDR, VL and VH regions, heavy chains and light chains may have the same sequence as provided herein.

[0160] The first HC may include charged paired permutations K392D and K409D, and the second heavy chain may include charged paired permutations E356K and D399K, or vice versa.

[0161] In a specific embodiment, the bispecific antibody of the present invention does not include the bispecific antibody, UIC2 DD HC :: Cetuximab KK HC :: MRK16 LC. This bispecific antibody includes a VH chain comprising the anti-MDR1 antibody, HCDR1-3 of UIC2. However, this antibody does not maintain binding to MDR1 as measured by FACS using 293T cells overexpressing MDR1. Refer to FIG. 31. The VH chain of UIC2 HC is as provided elsewhere in the present invention.

[0162] In a specific embodiment, the bispecific antibody comprises a second heavy chain comprising the second VH chain and the heavy chain constant region described herein. The heavy chain may comprise the sequence of the human IgG1 heavy chain constant region.

[0163] In a specific embodiment, the bispecific antibody molecule specifically binds to cells expressing both MDR1 and TAA and has a 2x higher affinity for cells expressing both MDR1 and TAA compared to cells expressing MDR1 or TAA.

[0164] In a specific embodiment, a bispecific antibody molecule can increase the sensitivity of cancer cells to treatment with a chemotherapy agent, and the half-maximum inhibitory concentration (IC50) of the chemotherapy agent is a VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA; and the IC50 of the chemotherapy agent when co-administered with the anti-MDR1 antibody containing the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK is at least 2 times (e.g., at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, or at least 30 times) lower than that of the chemotherapy agent when co-administered with the antibody. In certain embodiments, the anti-MDR1 antibody may be the 15D3 antibody described in U.S. Patent No. 5,959,084.

[0165] In a specific embodiment, a bispecific antibody molecule binds to MDR1 with an affinity at least 2 times lower (e.g., at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, or at least 30 times lower) than an anti-MDR1 antibody comprising: a VH chain with the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA; and a VL chain with the following sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK. In a specific embodiment, the anti-MDR1 antibody may be the 15D3 antibody described in U.S. Patent No. 5,959,084.

[0166] In a specific embodiment, a bispecific antibody molecule inhibits efflux by MDR1 when it binds to a cell expressing MDR1. The inhibition can reduce efflux by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or more compared to efflux by MDR1 in the absence of the bispecific antibody.

[0167] In a specific embodiment, the bispecific antibody molecule comprises an Fc domain modified to reduce or abolish the binding of the antibody to one or more Fcγ receptors. In a specific embodiment, the IgG1 Fc domain may have one or more of the substitutions L234A, L235A, P329G, and N297A / Q / G.

[0168] As summarized above, the present invention provides a multispecific antibody having a domain targeting a cell efflux pump and a domain targeting a cancer-associated antigen. The multispecific antibody comprises a multidrug resistance protein 1 (MDR1)-binding domain and a leukocyte surface antigen CD47-binding domain. The multispecific antibody of the present invention specifically binds to cells expressing both MDR1 and CD47.

[0169] Therefore, the multispecific antibody of the present invention targets both MDR1 and CD47. MDR1, also known as P-glycoprotein 1 (Pgp), is an energy-dependent efflux pump that causes reduced drug accumulation in multidrug-resistant cells, expressed from ATP-binding cassette subfamily B member 1 (ABCB1). CD47, also known as integrin-associated protein (IAP), is a transmembrane protein of the immunoglobulin superfamily encoded by the CD47 gene that binds to membrane integrins and also acts as a receptor for the ligands thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα). CD47 ligand binding can inhibit 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.

[0170] A schematic diagram of a specific embodiment of the multispecific antibody of the present invention is provided in FIG. 1A. As illustrated, the multispecific antibody (100) comprises an MDR1-binding domain (101) and a CD47-binding domain (102), and optionally comprises all or part of an Fc domain (103). As illustrated in FIG. 1B, in the presence of a cell (104) that expresses MDR1 (107) but has low or no CD47 expression, the multispecific antibody (100) has low affinity for the cell. Correspondingly, in the presence of a cell (105) that expresses CD47 (108) but has low or no MDR1 expression, the multispecific antibody (100) has low affinity for the cell. However, in the presence of cells (106) expressing both MDR1 (107) and CD47 (108), the multispecific antibody (100) has a high affinity for the cells.

[0171] Accordingly, the multispecific antibody of the present invention binds to cells expressing both MDR1 and CD47 with a higher affinity than to cells expressing only MDR1 or CD47. Correspondingly, the multispecific antibody of the present invention binds with a much reduced affinity when a low level of each second target is present, for example, compared to when both the first target and the second target are present at levels higher than low (e.g., average, normal, and / or high levels). In some embodiments, the affinity with which the target multispecific antibody binds to cells expressing both MDR1 and CD47 is twofold, e.g., 2.5fold, 3fold, 4fold, 5fold, 6fold, 7fold, 8fold, 9fold, 10fold, or higher than the affinity with which the target multispecific antibody binds to cells expressing MDR1 or CD47 (or at low levels of MDR1 or CD47).

[0172] In some embodiments, the target multispecific antibody can prevent the function of the cellular MDR1 protein when it binds to a cell expressing MDR1. Accordingly, the multispecific antibody of the present invention can suppress efflux by the MDR1 protein, for example, 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 efflux by MDR1 in the absence of the target multispecific antibody.

[0173] In some embodiments, the target multispecific antibody can prevent the function of the cellular CD47 protein when it binds to a cell expressing CD47. Accordingly, the multispecific antibody of the present invention can inhibit the binding of a CD47-ligand or CD47 binding partner to CD47, for example, the binding of the ligand / binding partner 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 the binding by CD47 in the absence of the target multispecific antibody.

[0174] The multispecific antibodies of the present invention are bispecific for at least MDR1 and CD47, and the configuration of the antibodies may differ. 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 partial fragments thereof, capable of binding to an epitope. With respect to the multispecific antibodies described herein, such antibodies may bind to at least two different epitopes present on two different target proteins. The number of different target proteins, and thus different epitopes, bound by the target multispecific antibodies may vary and may be two (i.e., bispecific), three (triplespecific), four, or more.

[0175] In some embodiments, the multispecific antibody of the present invention may comprise a common light chain. As used herein, the term “common light chain” generally refers to the use of two copies of the same light chain and their integration into a multispecific antibody. In other words, in the assembled multispecific antibody, the light chain will associate with the MDR1-specific heavy chain and a second copy of the same light chain will associate with the CD47-specific heavy chain.

[0176] The VH and VL regions can be further subdivided into a more conserved region called the "Framework Region (FR)" and an interspersed hypervariable region called the "Complementarity Determining Region (CDR)." The degrees of the FR and CDR are precisely defined (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1987) J. Mol. Biol. 196: 901-917). The VH may contain three CDRs and four FRs arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, VL may include three CDRs and four FRs arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0177] The VH or VL chain of the antibody may further include all or part of a heavy or light chain constant region to form a heavy or light chain immunoglobulin, respectively. In one embodiment, the antibody is a tetramer of two heavy chains and two light chains, and the heavy and light chains are connected to each other, for example, by disulfide bonds. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains include binding regions that interact with the antigen. The constant region of the antibody typically mediates the binding of the antibody to factors, including the first complement of the complement system and various cells of the host tissue and immune system. The term "antibody" includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM, and their subtypes. In some embodiments, the target antibody is an IgG isotype.

[0178] 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 many immunoglobulin variable region genes. The 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 by a kappa or lambda constant region at the C-terminus. The 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 by one of the aforementioned constant region genes, for example, gamma (encoding about 330 amino acids), at the C-terminus. In some embodiments, the target antibody comprises a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0179] In some embodiments, the target antibody does not include a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, but instead includes 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, the antigen-binding fragments are contained on separate polypeptide chains; in other embodiments, the antigen-binding fragments are contained within a single polypeptide chain.

[0180] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody capable of specifically binding to MDR1 or CD47 as described above. Examples of binding fragments include (i) a Fab fragment (a monovalent fragment comprising VL, VH, CL, and CH1 domains, e.g., consisting thereof); (ii) an F(ab')2 fragment (a divalent fragment comprising two Fab fragments connected by a disulfide bridge at a hinge region); (iii) an Fd fragment (containing VH and CH1 domains, e.g., consisting thereof); (iv) an Fv fragment (containing VH and VL domains of a single arm of the antibody, e.g., consisting thereof); (v) a dAb fragment (containing a VH domain, e.g., consisting thereof); (vi) an isolated CDR; (vii) single-stranded Fv (scFv) (comprising the VH and VL domains of a single arm of an antibody combined by a synthetic linker using recombinant means so that the VH and VL domains pair to form a monovalent molecule, e.g., composed of these); (viii) diabody (comprising two scFvs in which the VH and VL domains are combined so that the VH and VL domains do not pair to form a monovalent molecule, e.g., composed of these; each of the VH of the scFv pairs with the VL domain of the other scFv to form a divalent molecule).

[0181] In some embodiments, the target antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deiminized, or in vitro ( in vitro ) is a generated antibody. The terms “recombinant” or “modified” antibody are intended to include any antibody produced, expressed, generated, or isolated by recombinant means as used herein, e.g., (i) an antibody expressed using a recombinant expression vector transfected into a host cell; (ii) an antibody isolated from a recombinant combinational antibody library; (iii) an antibody isolated from an animal (e.g., mouse) genetically modified for a human immunoglobulin gene; or (iv) an antibody produced, expressed, generated, or isolated by any other means involving splicing of a human immunoglobulin gene sequence into another DNA sequence. Such recombinant antibodies include humanized, CDR-transplanted, chimeric, deiminized, and in vitro generated antibodies; and may optionally include an invariant region derived from a human germline immunoglobulin sequence.

[0182] The modified antibody may include a modified domain, for example, any antibody domain may be modified from its naturally occurring form. In some embodiments, the modified antibody may include a modified heavy chain, a modified Fc domain, and a modified CH2 and / or modified CH3 domain. In some cases, the modified Fc domain may utilize electrostatic steering effects, including, but not limited to, using the procedure described in, for example, Gunasekeran et al, (2010) Journal of Biological Chemistry 285, 19637-19646 (the full text of which is incorporated herein by reference). In some cases, the bispecific antibody is assembled through charge pair substitution in the CH3 domain, for example, one heavy chain is modified to contain K392D and K409D substitutions and the other heavy chain is modified to contain E356K and D399K substitutions. Charge pair-substituted chains can preferentially form heterodimers with each other. The numbering of amino acid substitutions follows the EU numbering system for HC.

[0183] In some cases, the antibody of the present invention comprises charge pair substitution. In some cases, the antibody of the present invention does not comprise charge pair substitution. In some cases, alternative means for promoting the preferential heterodimer formation of the desired chain may be used.

[0184] In some cases, the modified heavy chain may include a knob-into-hole modification. "Knob-into-hole" amino acid modification is a reasonable design strategy in antibody manipulation used for the heterodimerization of heavy chains during the production of multispecific antibodies, including bispecific IgG antibodies. For example, in incorporating the knob-into-hole strategy into a bispecific antibody made from two monoclonal antibodies of different specificities, amino acid modifications are manipulated to create 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 appear as a large amino acid, e.g., tyrosine (Y), while the hole can appear as a small amino acid, e.g., threonine (T). For example, the Knob-into-hole paired modification can produce 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, JB et al. Protein Eng. 9(7):617-2 (1996); and Merchant, AM et al. Nat. Biotechnol. 16(7):677-81 (1998) (the full text of which is incorporated herein). In antibodies produced from paired Knob-into-hole modified domains, the bispecific heterodimer will generally represent the major fraction.

[0185] As summarized above, the multispecific antibody of the present invention will include an MDR1-binding domain and a CD47-binding domain. These domains may vary and include epitopes bound by the domains, variable region arrangements and sequences, etc.

[0186] The target MDR1-binding domain 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 may vary, and for example, the MDR1 epitope may be in the range of 4 aa or less to 12 aa or less, and is formed by a polypeptide having adjacent extensions of an MDR1 sequence such as, for example, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 4 aa to 10 aa, 5 aa to 10 aa, 6 aa to 10 aa, 4 aa to 8 aa, 5 aa to 8 aa, 6 aa to 8 aa, etc.

[0187] In some embodiments, the MDR1 epitope is an adjacent elongation of the MDR1 sequence, for example, the human MDR1 sequence:

[0188]

[0189]

[0190] Macaca fascicularis ) (Crab-eating monkey) hierarchy:

[0191]

[0192] The target MDR1-binding domain exhibits high affinity binding to MDR1. For example, the target MDR1-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 -12 It binds to MDR1 with an affinity greater than M. The target MDR1-binding domain is approximately 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 to an epitope present on MDR1 with an affinity greater than M.

[0193] The target MDR1-binding domain does not substantially represent binding to any epitope formed by amino acids within other related but sequence-different proteins, e.g., an EP that is related but sequence-different. Any binding of the target MDR1-binding domain to an epitope formed by amino acids within a related but sequence-different protein is generally a non-specific binding with substantially lower affinity than the specific binding of the MDR1-binding domain to the epitope on MDR1. Substantially lower affinity is generally at least 2, 3, 5, 10, 50, 100, 500, or 1000 times lower affinity.

[0194] The target MDR1-binding domain can reduce the transport of molecules through the MDR1 transporter. For example, the target MDR1-binding domain 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.

[0195] In some embodiments, the target antibody comprises a) i. a CDR1 region having the same amino acid sequence as the heavy chain CDR1 region of RYTMS or containing the heavy chain CDR1 region of RYTMS; ii. a CDR2 region having the same amino acid sequence as the heavy chain CDR2 region of TISSGGGNTYYPDSVKG or containing the heavy chain CDR2 region of TISSGGGNTYYPDSVKG; and iii. a heavy chain variable domain having the same amino acid sequence as the heavy chain CDR3 region of YGAGDAWFAY or containing the heavy chain CDR3 region of YGAGDAWFAY. These CDR1-3 regions are the VH chain of the 15D3 antibody with the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA or the humanized version of this VH chain with the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Here X 2 is N, Q, or S) exists in. These CDRs are based on Kabat nomenclature.

[0196] In some embodiments, the target antibodies are CDR1 (RYTMS), CDR2 (TISSGGG X 2 TYYPDSVKG (here X 2 A heavy chain variable region comprising 1, 2, or 3 heavy chain CDRs having amino acid sequences selected from one or more of CDR3 (YGAGDAWFAY)), and CDR3 (YGAGDAWFAY); and a FR region comprising mammalian sequences, for example, rodents, non-human primates, and human sequences (e.g., encoded by each heavy chain FR-coding sequence). For example, in some embodiments, the target antibody comprises a heavy chain variable region comprising the following in order from the N-terminus to the C-terminus: human heavy chain FR1; CDR1 containing the amino acid sequence RYTMS; human heavy chain FR2; CDR2 containing the amino acid sequence TISSGGGNTYYPDSVKG; human heavy chain FR3; CDR3 containing the amino acid sequence YGAGDAWFAY; and human heavy chain FR4.

[0197] The target antibody may comprise a heavy chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100% identical to the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS. The target antibody may comprise a heavy chain variable region comprising one, two, or three heavy chain complementarity determining regions (CDRs) having amino acid sequences selected from one or more of CDR1 (RYTMS), CDR2 (TISSGGGNTYYPDSVKG), and CDR3 (YGAGDAWFAY). In a specific embodiment, the VH chain is humanized to include a human framework region, and the humanized VH chain may comprise an amino acid sequence identical to the following sequence by 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100%: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Here X 2 is N, Q, or S).

[0198] The target 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 may vary, and, for example, the CD47 epitope may be in the range of 4 aa or less to 12 aa or less, and is formed by a polypeptide having adjacent extensions of the CD47 sequence, such as, for example, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 4 aa to 10 aa, 5 aa to 10 aa, 6 aa to 10 aa, 4 aa to 8 aa, 5 aa to 8 aa, 6 aa to 8 aa, etc.

[0199] In some embodiments, the CD47 epitope is an adjacent extension of the CD47 sequence, for example, the human CD47 sequence:

[0200] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGI KTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE; or a rodent CD47 sequence, such as a mouse CD47 sequence:

[0201] MWPLAAALLLGSCCCGSAQLLFSNVNSIEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTTTDQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSWFSPNEKILIVIFPILAILLFWGKFGILTLKYKSSHTNKRIILLLVAGLVLTVIVVVGAILLIPGEKPVKNASGLGLIVISTGILILLQYNVFMTAFGMTSFTIAILITQVLGYVLALVGLCLCIMACEPVHGPLLISGLGIIALAELLGLVYMKFVASNQRTIQPPRNR, or non-human primate sequences, e.g., Sumatran orangutan ( Pongo abelii ) (Sumatra Orangutan) Ranking:

[0202] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGI KTLKYRSGGMDEKTIALLVAGLIITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLNSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE, or Bengal monkey ( Macaca mulatta ) (Rhesus monkey) hierarchy:

[0203] For MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE, etc., 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 It can be formed by a polypeptide having

[0204] The target CD47-binding domain exhibits high affinity binding for CD47. For example, the target 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 -12 It binds to CD47 with an affinity greater than M. The target CD47-binding domain is approximately 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 -12It binds to an epitope on CD47 with an affinity greater than M.

[0205] The target CD47-binding domain does not substantially represent binding to any epitope formed by amino acids within other related but sequence-different proteins, such as an immune checkpoint marker that is related but sequence-different. Any binding of the target CD47-binding domain to an epitope formed by amino acids within a related but sequence-different protein is generally a non-specific binding with substantially lower affinity than the specific binding of the CD47-binding domain to the epitope on CD47. Substantially lower affinity is generally at least 2, 3, 5, 10, 50, 100, 500, or 1000 times lower affinity.

[0206] The target CD47-binding domain can reduce the binding of CD47-binding partners to CD47, e.g., thrombospondin-1 (TSP-1), signal regulatory protein alpha (SIRPα), and integrins (e.g., integrin avb3). For example, the target 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.

[0207] In some embodiments, the target antibody comprises a) i. a CDR1 region having the same amino acid sequence as the CDR1 region of NYNMH or containing the CDR1 region of NYNMH; ii. a CDR2 region having the same amino acid sequence as the heavy chain CDR2 region of TIYPGNDDTSYNQKFKD or containing the heavy chain CDR2 region of TIYPGNDDTSYNQKFKD; and iii. a heavy chain variable domain having the same amino acid sequence as the heavy chain CDR3 region of GGYRAMDY or containing the CDR3 region of GGYRAMDY. These CDR1-3 regions are present on the VH chain of the 5F9 antibody with the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS. These CDRs are based on the Kabat nomenclature.

[0208] The target antibody may include a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100% identical to the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS. The target antibody may include a heavy chain variable region comprising 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).

[0209] In some embodiments, the target antibody comprises a heavy chain variable region comprising one, two, or three heavy chain CDRs having amino acid sequences selected from one or more of CDR1 (NYNMH), CDR2 (TIYPGNDDTSYNQKFKD), and CDR3 (GGYRAMDY); and an FR region which is a mammalian, e.g., rodent, non-human primate, or human sequence (e.g., encoded by each heavy chain FR-coding sequence). For example, in some embodiments, the target antibody comprises a heavy chain variable region comprising the following in order from the N-terminus to the C-terminus: human heavy chain FR1; CDR1 containing the amino acid sequence NYNMH; human heavy chain FR2; CDR2 amino acid sequence containing the amino acid sequence TIYPGNDDTSYNQKFKD; human heavy chain FR3; CDR3 containing GGYRAMDY; and human heavy chain FR4.

[0210] The target antibody is an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, e.g., 100% identical to the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK or a humanized version of this sequence having the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGX 1 TYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (Here X 1 It includes a light chain variable region containing N, Q, or S).

[0211] The target antibody is CDR1 (RSSQSIVHSTG X 1 TYLEW) (Here X 1It may include a light chain variable region comprising a light chain CDR having the amino acid sequence presented in CDR1 (RSSQSLLHSDGFDYLNW), CDR2 (ALSNRASG), and CDR3 (MZALQAPITF), where N, Q, or S is present. In some cases, such a light chain variable region may be utilized in the common light chain of a target multispecific antibody.

[0212] In some embodiments, the target antibody is CDR1 (RSSQSIVHSTG X 1 TYLEW) (Here X 1 A light chain variable region comprising a light chain CDR having the amino acid sequence presented in CDR1 (RSSQSLLHSDGFDYLNW), CDR2 (ALSNRASG), and CDR3 (MZALQAPITF), wherein is N, Q, or S); and a FR region that is a mammalian, e.g., rodent, non-human primate, or human sequence (e.g., encoded by the respective light chain FR-coding sequence). For example, in some embodiments, the target antibody comprises a light chain variable region comprising the following in the order from the N-terminus to the C-terminus: human light chain FR1; amino acid sequence RSSQSIVHSTGX 1 CDR1 containing TYLEW (where X 1 is N, Q or S); human light chain FR2; CDR2 containing the amino acid sequence KISNRFSG; human light chain FR3; CDR3 containing the amino acid sequence presented in FQASHFPRTF; and human light chain FR4.

[0213] In some embodiments, the target antibody optionally comprises a heavy chain FR1 region; a CDR1 containing the amino acid sequence RYTMS; a heavy chain FR2 region; a CDR2 containing the amino acid sequence TISSGGG(N / S / Q)TYYPDSVKG; a heavy chain FR3 region; a CDR3 containing the amino acid sequence YGAGDAWFAY; and a heavy chain FR4 region; and optionally a heavy chain FR1 region; a CDR1 containing the amino acid sequence NYNMH; a heavy chain FR2 region; a CDR2 containing the amino acid sequence TIYPGNDDTSYNQKFKD; a heavy chain FR3 region; a CDR3 containing the amino acid sequence GGYRAMDY; and a heavy chain FR4 region. In some of these embodiments, each FR region is a mammalian FR region, e.g., a human FR region.

[0214] In some embodiments, the target antibody comprises the heavy chain sequence and light chain FR1 region described in the previous paragraph; CDR1 comprising the amino acid sequence RSSQSIVHSTGNTYLEW or RSSQSLLHSDGFDYLNW; light chain FR2 region; CDR2 comprising the amino acid sequence KISNRFSG or ALSNRASG; light chain FR3 region; CDR3 comprising the amino acid sequence FQASHFPRTF or MZALQAPITF; and optionally also comprises the light chain FR4 region.

[0215] In some embodiments, the target antibody is an anti-MDR1 heavy chain sequence comprising a charge-to-charge swap (KK) variant having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,Anti-CD47 heavy chain sequence containing a charge-to-charge exchange (DD) variant with the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, and anti-MDR1 light chain sequence having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. In some cases, the target antibody may include an alternative heterodimer Fc pairing strategy.

[0216] Charge-to-charge exchange variants represent substitutions 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. Charge-paired substituent chains prefer to form heterodimers with each other. The numbering of amino acid substitutions follows the EU numbering system for Ig HC.

[0217] Monospecific bivalent antibodies and bispecific antibodies

[0218] An antibody that may be a monospecific bivalent antibody or a bispecific antibody derived therefrom is also provided herein.

[0219] In a specific embodiment, the antibody of the present invention comprises a variable light chain (VL) comprising a light chain CDR (LCDR) of the VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (here X 1 is N, Q, or S); and a variable heavy chain (VH) containing a heavy chain CDR (HCDR) of the VH chain with the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (where 2 It includes N, Q, or S).

[0220] In a specific embodiment, the VL chain comprises an LCDR of the VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK; The VH chain comprises the HCDR of the VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or the VL chain comprises the LCDR of the VL chain having the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; The VH chain comprises the HCDR of the VH chain having the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or the VL chain comprises the LCDR of the VL chain having the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; The VH chain contains the LCDR of the VH chain with the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0221] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGNTYLE, RSSQSIVHSTGQTYLE, or RSSQSIVHSTGSTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT.

[0222] In a specific embodiment, HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGNTYYPDSVKG, TISSGGGQTYYPDSVKG, or TISSGGGSTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0223] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGNTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGNTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0224] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGQTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGQTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0225] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGSTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGSTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0226] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGNTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGQTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY, or LCDR1 comprises the following sequence: RSSQSIVHSTGNTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGSTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0227] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGQTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGNTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0228] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGSTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGNTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0229] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGSTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGQTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0230] In a specific embodiment, LCDR1 comprises the following sequence: RSSQSIVHSTGNTYLE, LCDR2 comprises the following sequence: KISNRFS, and LCDR3 comprises the following sequence: FQASHFPRT; HCDR1 comprises the following sequence: RYTMS, HCDR2 comprises the following sequence: TISSGGGQTYYPDSVKG, and HCDR3 comprises the following sequence: YGAGDAWFAY.

[0231] In a specific embodiment, the antibody is a monospecific bivalent antibody that specifically binds to MDR-1. In a specific embodiment, 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 VL chain having the following amino acid sequence:

[0232] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK;

[0233] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK; or

[0234] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK.

[0235] In a specific embodiment, amino acid residues different from the VL chain sequence or VH chain sequence are located in the framework region.

[0236] In a specific embodiment, 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 a VH chain having the following amino acid sequence:

[0237] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS;

[0238] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS; or

[0239] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0240] In a specific embodiment, the antibody is a bispecific antibody containing a VL chain as a common light chain.

[0241] In a specific embodiment, the bispecific antibody comprises an MDR-1 binding domain and a tumor-associated antigen (TAA) binding domain, wherein the MDR-1 binding domain and the TAA binding domain each comprise LCDR1-3 of the VL chain.

[0242] In a specific embodiment, TAA is CD47 and the TAA binding domain contains the HCDR of the VH chain having the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0243] In a specific embodiment, the VH chain of the CD47 binding domain comprises HCDR1: NYNMH containing the following sequence, HCDR2: TIYPGNDDTSYNQKFKD containing the following sequence, and HCDR3: GGYRAMDY containing the following sequence.

[0244] In a specific embodiment, the antibody of the present invention comprises a variable light chain (VL) comprising a light chain CDR (LCDR) of the VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (here X 1is N, Q, or S); and a variable heavy chain (VH) comprising a heavy chain CDR (HCDR) of the VH chain with the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS

[0245] In a specific embodiment, the VH chain comprises HCDR1: NYNMH containing the following sequence, HCDR2: TIYPGNDDTSYNQKFKD containing the following sequence, and HCDR3: GGYRAMDY containing the following sequence.

[0246] In a specific embodiment, the antibody is a monospecific bivalent antibody that specifically binds to CD47. In another embodiment, the antibody is a bispecific antibody that binds to CD47 and MRD-1.

[0247] In a specific embodiment, a monospecific bivalent antibody that specifically binds to MDR-1 and a monospecific bivalent antibody that specifically binds to CD47 may be used in a method for treating cancer in a subject as described herein, and the method may involve the step of co-administering two antibodies to the subject in an amount effective for treating cancer. In a specific embodiment, the method may further involve the step of administering a chemotherapy agent to the subject.

[0248] The region and / or chain of the target antibody may or may not be bound by one or more linker regions. If present, the linker region may have a length of about 5 amino acids to about 50 amino acids, for example, a length of 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.

[0249] Linkers suitable for use in target antibodies include "flexible linkers." If present, the linker molecule is generally long enough to allow some flexible movement between linked regions. Linker molecules are typically about 6 to 50 atoms long. The linker molecule may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, dibasic acids, amino acids, or combinations thereof. Other linker molecules capable of binding to polypeptides may be used in consideration of the present invention.

[0250] A suitable linker can be easily selected and may be of a suitable different length, e.g., 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, e.g., 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids or 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0251] The flexible linker of the example is a glycine polymer (G) n , glycine-serine polymer (e.g., (GS) n , GSGGSn (Sequence No.: / / ) and GGGS n (Sequence number: / / ), where n is an integer of at least 1), includes 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 not relatively constructed and can therefore act as a neutral tether between the components. Glycine polymers are of particular interest because glycine accesses much more phi-psi space than alanine and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. See 11173-142 (1992). The flexible linkers of the examples include, but are not limited to, GGSG (sequence number:: / / ), GGSGG (sequence number:: / / ), GSGSG (sequence number: / / ), GSGGG (sequence number: / / ), GGGSG (sequence number: / / ), GSSSG (sequence number: / / ), etc. Those skilled in the art will acknowledge that the design of a peptide conjugated to any of the elements described above may include a fully or partially flexible linker so that the linker may include a flexible linker as well as one or more parts that impart a less flexible structure.

[0252] In some embodiments, the target antibody is “humanized.” The term “humanized antibody” refers to an antibody comprising at least one chain comprising a variable region framework residue from a substantially human antibody chain (recipient immunoglobulin or antibody) and at least one CDR from a substantially non-human antibody (e.g., rodent (e.g., mouse antibody), non-human primate, etc.) (recipient immunoglobulin or antibody). References should be made to Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 10033 (1989), U.S. Patent No. 5,530,101, U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761, WO 90 / 07861, and U.S. Patent No. 5,225,539. The constant region(s) may also, if present, be derived substantially or entirely from human immunoglobulin. In some embodiments, the target antibody comprises one or more MDR1 CDRs, one or more CD47 CDRs, and one or more FR regions from a human antibody. Methods for producing humanized antibodies are known in the art. For example, one may refer to U.S. Patent No. 7,256,273.

[0253] The substitution of a mouse CDR into a human variable domain framework can maintain accurate spatial orientation, for example, if the human variable domain framework adopts a stereostructure identical or similar to that of the mouse variable framework from which the CDR is derived. This can be achieved by obtaining the human variable domain from a human antibody in which the framework sequence exhibits 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 may be derived from identical or different human antibody sequences. The human antibody sequence may be the sequence of a naturally occurring human antibody or a common sequence of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

[0254] Once the complementation determining regions of the mouse donor immunoglobulin and the appropriate human recipient immunoglobulin are identified, the next step is to determine, if present, whether residues from these components should be substituted to optimize the properties of the resulting humanized antibody. In general, the substitution of human amino acid residues with mouse amino acid residues should be minimized, as the introduction of mouse residues increases the risk that the antibody will induce a human-anti-mouse-antibody (HAMA) response in humans. Immunological response determination methods recognized in the art may be performed to monitor the HAMA response in specific patients or during clinical trials. Immunogenicity assessments of the humanized antibody administered to the patient may be provided at the beginning of administration and throughout the course of the therapy. The HAMA response is measured in the patient's serum sample by detecting antibodies against the humanized therapeutic agent, for example, using methods known to those skilled in the art, including surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA assays. In many embodiments, the target humanized antibody does not substantially induce a HAMA response in human subjects.

[0255] Specific amino acids from human variable region framework residues are selected for substitution based on their potential influence on CDR stereostructure and / or binding to antigens. The unnatural arrangement of rat CDR regions with human variable region domains can cause unnatural stereostructure restrictions, which lead to a loss of binding affinity unless corrected by the substitution of specific amino acid residues.

[0256] The selection of amino acid residues for substitution can be determined in part by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, a molecular model is generated starting from a decoded structure for its immunoglobulin chain or domain. The chain to be modeled is compared with the chain or domain of the decoded three-dimensional structure for amino acid sequence similarity, indicating that the highest sequence similarity is selected as the starting point for constructing the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, and preferably those sharing at least 60%, 70%, 80%, 90%, or higher sequence identity are selected for modeling. The decoded starting structure is modified to allow for a difference between the actual amino acids of the immunoglobulin chain or domain being modeled and the amino acids of the starting structure. The modified structure is assembled into synthetic immunoglobulin. Finally, the model is improved by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0257] 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 were 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 framework residues constitute structural loop residues defined by Kabat, as defined above, and by Chothia, as defined above, amino acids present in mouse antibodies can be selected for substitution with humanized antibodies. Residues "adjacent to the CDR region" include amino acid residues located immediately adjacent to one or more of the CDRs of the major sequence of the humanized immunoglobulin chain, for example, the CDR defined by Kabat, or the CDR defined by Chothia (see, for example, Chothia and Lesk JMB 196:901 (1987)). These amino acids are likely to interact with amino acids, particularly in the CDR, and if selected from the recipient, are likely to twist the donor CDR and reduce affinity. Furthermore, adjacent amino acids can interact directly with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to maintain all antigen contacts that provide affinity to the original antibody.

[0258] In some embodiments, the target antibody comprises scFv multimers. For example, in some embodiments, the target antibody is a scFv dimer (e.g., comprising two tandem scFvs (scFv2)), a scFv trimer (e.g., comprising three tandem scFvs (scFv3)), a scFv tetramer (e.g., comprising four tandem scFvs (scFv4)), or a multimer of more than four scFvs (e.g. tandem). The scFv monomers may be linked tandem via a linker of lengths ranging from about 2 amino acids to about 10 amino acids, for example, lengths of 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. Suitable linkers are, for example, (Gly) x It includes, where x is an integer from 2 to 10. Other suitable linkers are those discussed above. In some embodiments, each scFv monomer in the target scFV multimer is humanized as described above.

[0259] In some embodiments, the target antibody comprises an immutable region of the immunoglobulin (e.g., an Fc region). The Fc region may be a human Fc region, if present. If an immutable region is present, the antibody may contain both a light chain and a heavy chain immutable region. Suitable heavy chain immutable regions include CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include antibodies having all types of immutable regions, including IgM, IgG, IgD, IgA, and IgE, and any isotype, such as IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is the human isotype IgG1 Fc. The light chain immutable region may be lambda or kappa. The target antibody (e.g., a target humanized antibody) may comprise a sequence from more than one class or isotype. The antibody may be expressed as a tetramer containing two light chains and two heavy chains, as distinct heavy chains and light chains, as Fab, Fab' F(ab')2, and Fv, or as a single-chain antibody in which the heavy chain and light chain variable domains are connected through a spacer.

[0260] In some embodiments, the target antibody includes a free thiol (-SH) group at the carboxyl terminus, and the free thiol group can be used to attach the antibody to a second polypeptide (e.g., another antibody containing the target antibody), a scaffold, a carrier, etc.

[0261] The target antibody may be covalently bonded to a second moiety (e.g., lipids, polypeptides other than the target antibody, synthetic polymers, carbohydrates, etc.) using, for example, glutaraldehyde, a homobifunctional cross-linker, or a heterobifunctional cross-linker. Glutaraldehyde cross-links to polypeptides via amino moiety. Homobifunctional cross-linkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfidyl reactive cross-linkers) may be used in a one-step reaction procedure in which the linker is added to a solution containing a mixture of polypeptides linked with two or more identical reactive moietys. Homobifunctional NHS esters and imidoesters cross-link to amine-containing polypeptides. At a weakly alkaline pH, imido esters react only with primary amines to form imidoamides, and the total charge of the cross-linked polypeptide is not affected. Homomorphic difunctional sulfidyl-reactive cross-linkers include bismaleimidehexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propinoamidobutane (DPDPB).

[0262] Composition and preparation

[0263] The present invention provides a composition comprising a target antibody. In addition to the target antibody, the target antibody composition may comprise one or more of the following: a salt, e.g., NaCl, MgCl2, KCl, MgSO4, etc.; a buffer, e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizer; a detergent, e.g., a non-ionic detergent, e.g., Tween-20, etc.; a protease inhibitor; glycerol; etc.

[0264] The compositions of the present invention also include pharmaceutical compositions comprising the multispecific antibodies described herein. Generally, the formulation comprises an effective amount of the target antibody. "Effective amount" means a dosage sufficient to produce the desired result, e.g., a reduction in the cancer of the subject, a reduction in the growth rate of the cancer of the subject, an improvement in the symptoms of the cancer, etc. Generally, the desired result is, compared to a control group, at least a reduction in the symptoms of the cancer, a reduction in the growth of the cancer, a reduction in the size of the cancer, etc. The target antibody may be delivered or formulated in a manner to bypass the blood-brain barrier. In some cases, the antibody may include a delivery enhancer, and if such enhancer can facilitate crossing of the blood-brain barrier, it includes increased permeability allowing, e.g., efficient transdermal delivery, etc. Useful delivery enhancers include, for example, cerepot, legadenosone, borneol, puerarin, propylene glycol, oleic acid, azone, N-methylpyrrolidone, Tween 80, limonene, lipid-based nanoparticles (NP), liposomes, niosomes, transferosomes, ethosomes, dendrimers, micelle NPs, polymer nanostructures, metallic nanostructures, magnetic nanostructures, recombinant human hyaluronidase, etc., but are not limited thereto.

[0265] In the target method, the target antibody may be administered to a host using any convenient means capable of producing the desired therapeutic or diagnostic effect. Thus, the agent may be included in various formulations for therapeutic administration. More specifically, the target antibody may be formulated into a pharmaceutical composition by combination with a suitable pharmaceutically acceptable carrier or diluent, and may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0266] In pharmaceutical formulations, the target antibody may be administered with pharmaceutically acceptable excipients, or used alone or in appropriate association with other pharmaceutically active compounds, as well as in combination with them. The following methods and excipients are merely examples and are not limiting.

[0267] For oral formulations, the target antibody may be used alone or in combination with suitable additives for making tablets, powders, granules, or capsules, e.g., conventional additives, e.g., lactose, mannitol, corn starch, or potato starch; binders, e.g., crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; degrading agents, e.g., corn starch, potato starch, or sodium carboxymethylcellulose; lubricants, e.g., talc or magnesium stearate; and, if desired, diluents, buffers, wetting agents, preservatives, and flavoring agents. In some cases, oral delivery of the antibody may be enhanced through the formation of a complex of the antibody with a suitable hydrogel.

[0268] Target antibodies may be formulated into injectable preparations by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents, such as vegetable or other similar oils, synthetic aromatic acid glycerides, higher aromatic acids, or propylene glycol esters; and, if desired, in conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

[0269] A pharmaceutical composition comprising a target antibody is prepared by mixing an antibody of a desired degree of purity with a selective, physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer, and / or isotonic agent. The acceptable carrier, excipient, and / or stabilizer is non-toxic to the recipient at the dose and concentration used and comprises: buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, e.g., ascorbic acid, glutathione, cysteine, methionine, and citric acid; and preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof). Amino acids, e.g., 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, e.g., gelatin or serum albumin; chelating agents, e.g., EDTA; sugars, e.g., trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants, e.g., Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0270] Pharmaceutical compositions may be in liquid form, lyophilized form, or liquid form restored from lyophilized form, and lyophilized preparations must be restored to a sterile solution before administration. The standard procedure for restoring a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however, a solution containing an antibacterial agent may be used in the production of pharmaceutical compositions for parenteral administration; and also refer to Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0271] The antibody concentration of the example in the target pharmaceutical composition may be in the range of about 1 mg / mL to about 200 mg / mL or about 50 mg / mL to about 200 mg / mL, or about 150 mg / mL to about 200 mg / mL.

[0272] Aqueous formulations of the antibody may be prepared in a pH-buffered solution at a pH range, for example, about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers, and other organic acid buffers. The buffer concentration may be, for example, about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on the desired properties of the buffer and formulation.

[0273] Isotonic agents may be included in antibody formulations to control the intensity of the formulation. Examples of isotonic agents include any component from the group of sodium chloride, potassium chloride, glycerin, and amino acids, sugars, as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution having the same intensity as some other solution being compared, e.g., physiological salt solution or serum. Isotonic agents may be used in amounts of about 5 mM to about 350 mM, e.g., 100 mM to 350 nM.

[0274] Surfactants may also be added to the antibody formulation to reduce aggregation of the formulated antibody and / or to minimize particle formation in the formulation and / or to reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan-fatty acid esters include 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 trade names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trade name Brij™. The concentration of examples of surfactants may be in the range of about 0.001% to about 1% w / v.

[0275] Antifreeze agents may also be added to protect active ingredients (e.g., proteins) that are unstable against destabilizing conditions during the freeze-drying process. For example, known antifreeze agents include sugars (e.g., glucose and sucrose); polyols (e.g., mannitol, sorbitol, and glycerol); and amino acids (e.g., alanine, glycine, and glutamic acid). Antifreeze agents may be included in an amount of about 10 mM to 500 nM.

[0276] In some embodiments, the target formulation comprises the target antibody and one or more of the identified agents (e.g., surfactants, buffers, stabilizers, isotonic agents) and essentially does not contain one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is included in the formulation at a concentration ranging, for example, about 0.001 to about 2% (w / v).

[0277] For example, the target formulation may be a liquid or lyophilized formulation suitable for parenteral administration and may comprise about 1 mg / mL to about 200 mg / mL of the target antibody; about 0.001% to about 1% of at least one surfactant; about 1 mM to about 100 mM of the buffer; optionally about 10 mM to about 500 mM of the stabilizer; and about 5 mM to about 305 mM of the isotonic agent; and have a pH of about 4.0 to about 7.0.

[0278] As another example, the target parenteral preparation is a liquid or lyophilized preparation comprising about 1 mg / mL to about 200 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5.

[0279] As another example, the target parenteral formulation comprises 1) a lyophilized formulation containing 15 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; having a pH of 5.5; or 2) a lyophilized formulation containing 75 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; having a pH of 5.5; or 3) a lyophilized formulation containing 75 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; having a pH of 5.5; or 4) 75 mg / mL of the target antibody; 0.04% Tween 20 w / v; 6) comprising a lyophilized preparation containing 20 mM L-histidine; and 250 mM trehalose; having a pH of 5.5; or 6) comprising a lyophilized preparation containing 75 mg / mL of target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; having a pH of 5.5.

[0280] As another example, the target parenteral formulation is 1) a liquid formulation containing 7.5 mg / mL of the target antibody; 0.022% Tween 20 w / v; 120 mM L-histidine; and 250 mM 125 mM sucrose; having a pH of 5.5; or 2) a liquid formulation containing 37.5 mg / mL of the target antibody; 0.02% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose; having a pH of 5.5; or 3) a liquid formulation containing 37.5 mg / mL of the target antibody; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose; having a pH of 5.5; or 4) 37.5 mg / mL of the target antibody; 0.02% Tween 20 w / v; A liquid formulation containing 10 mM L-histidine and 125 mM trehalose; having a pH of 5.5; or 5) a liquid formulation containing 37.5 mg / mL of the target antibody; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM trehalose; having a pH of 5.5; or 6) a liquid formulation containing 5 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; having a pH of 5.5; or 7) a liquid formulation containing 75 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol; having a pH of 5.5; or 8) a liquid formulation comprising 75 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride; having a pH of 5.5; or 9) a liquid formulation comprising 150 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; having a pH of 5.5; or 10) 150 mg / mL of the target antibody; 0.A liquid formulation comprising 0.2% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol; having a pH of 5.5; or 11) a liquid formulation comprising 150 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride; having a pH of 5.5; or 12) a liquid formulation comprising 10 mg / mL of the target antibody; 0.01% Tween 20 w / v; 20 mM L-histidine; and 40 mM sodium chloride; having a pH of 5.5.

[0281] The target antibody may be used in aerosol formulations administered by inhalation. The target antibody may be formulated with an acceptable pressurized propellant, such as dichlorodifluoromethane, propane, nitrogen, etc.

[0282] In addition, the target antibody can be prepared as a suppository by mixing it with various bases, such as emulsifying bases or water-soluble bases. The target antibody can be administered rectally through the suppository. The suppository may contain a vehicle such as cocoa butter, carbowax, and polyethylene glycol, which melts at body temperature but solidifies at room temperature.

[0283] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, and each dosage unit, e.g., teaspoonful, tablespoonful, tablet, or suppository, contains a predetermined amount of a composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the target antibody in the composition as a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier.

[0284] The term “unit dosing form”, as used herein, refers to physically distinct units suitable as a single dosing dose for human and animal subjects, each unit containing a predetermined amount of the compound of the present invention calculated to be sufficient to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier, or vehicle. Specifications for target antibodies may vary depending on the specific antibody used, the effect achieved, and the pharmacokinetics associated with each antibody in the host.

[0285] Other modes of administration will also be used with the present invention. For example, the target antibody may be formulated in suppositories, and in some cases, in aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories may be formed from a mixture containing an active ingredient in the range of about 0.5% to about 10% (w / w), for example, about 1% to about 2%.

[0286] The intranasal formulation will comprise a standard vehicle that does not cause irritation to the nasal mucosa and does not significantly interfere with ciliary body function. Diluents, such as water, aqueous saline, or other known substances, may be used in conjunction with the present invention. The intranasal formulation may also contain, but are not limited to, preservatives such as chlorobutanol and benzalkonium chloride. Surfactants may be present to enhance the absorption of the target protein by the nasal mucosa.

[0287] The target antibody may be administered as an injectable formulation. Typically, the injectable composition is prepared as a liquid solution or suspension; a solid form suitable for a solution in a liquid vehicle or suspension before injection may also be prepared. The formulation may also be emulsified, or the antibody may be encapsulated in a liposomal vehicle.

[0288] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or combinations thereof. In addition, if desired, the vehicle may contain small amounts of auxiliary substances such as wetting agents, emulsifying agents, or pH buffers. The actual method of manufacturing such dosage forms is known or will be obvious to those skilled in the art. For example, refer to Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. In any case, the composition or formulation administered will contain a sufficient amount of the target antibody to achieve the desired state in the subject being treated.

[0289] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Furthermore, pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, intestinal regulators, stabilizers, humectants, etc., are readily available to the public.

[0290] In some embodiments, the target antibody is formulated in a controlled-release formulation. Snustained-release formulations may be prepared using methods widely known in the art. Suitable examples of snustained-release formulations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody in which the matrix is ​​in the form of a molded body, e.g., a film or a microcapsule. Examples of snustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactide, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Possible loss of biological activity and possible change in the immunogenicity of the antibody contained in the snustained-release formulation can be prevented by using appropriate additives, by controlling the water content, and by developing a specific polymer matrix composition.

[0291] Within the scope of the present invention, controlled release may be taken to mean any one of many extended-release dosage forms. For the purposes of the present invention, the following terms may be considered substantially equivalent to controlled release: continuous release, controlled release, delayed release, depot, progressive release, long-term release, programmed release, extended release, proportional release, extended release, reservoir, retard, slow release, intervald release, sustained-release, time coat, periodic release, delayed action, sustained action, layered-time action, long-acting action, sustained action, repeated action, slow action, sustained-release action, sustained-release drug, and extended release. Further discussion of these terms may be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0292] Dosage

[0293] The appropriate dosage may be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is widely known in the medical field, the dosage for any single patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, the patient's gender, the time of year, the route of administration, general health status, and other drugs administered concurrently. The target antibody may be administered in amounts of 1 ng / kg body weight to 20 mg / kg body weight per dose, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 0.5 mg / kg body weight to 5 mg / kg body weight; however, doses lower or higher than the ranges in this example are conceived, particularly in consideration of the factors mentioned above. If the administration method is a continuous infusion, it may also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[0294] Those skilled in the art will readily acknowledge that dose levels may vary depending on the function of specific antibodies, the severity of symptoms, and the subject's sensitivity to side effects. The preferred dosage for a given compound can be easily determined by those skilled in the art by various means.

[0295] Administration route

[0296] Target antibodies are in vivo and in vitro ( ex vivo The drug is administered to an individual using any available method and route suitable for drug delivery, including systemic and local administration routes as well as the ) method.

[0297] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical, intravenous, intra-arterial, rectal, nasal, oral, and other enteric and parenteral routes of administration. Routes of administration may be combined if desired, or adjusted according to the antibody and / or desired effect. The target antibody composition may be administered as a single dose or multiple doses. In some embodiments, the target antibody composition is administered orally. In some embodiments, the target antibody composition is administered via the inhalation route. In some embodiments, the target antibody composition is administered intranasally. In some embodiments, the target antibody composition is administered topically. In some embodiments, the target antibody composition is administered intracranially. In some embodiments, the target antibody composition is administered intravenously.

[0298] The agent may be administered to a host using any available conventional method and route suitable for conventional drug delivery, including systemic or local routes. Generally, the routes of administration considered in the present invention include, but are not limited to, enteral, parenteral, or inhalation routes.

[0299] Routes of parenteral administration other than inhalation include, but are not limited to, local, transdermal, subcutaneous, intramuscular, intraorbital, intrasacral, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration may be performed to affect systemic or local delivery of the target antibody. If systemic delivery is desired, administration typically involves local or mucosal administration of the pharmaceutical preparation that is invasive or absorbed systemically.

[0300] Target antibodies can also be delivered to the subject by enteral administration. Routes of enteral administration include, but are not necessarily limited to, oral and rectal delivery (e.g., using suppositories).

[0301] Treatment refers to the improvement of symptoms associated with a pathological condition that causes harm to the host, and improvement is used in a broad sense to indicate a reduction in the magnitude of symptoms, for example, variables related to the pathological condition being treated, such as cancer and / or cancer growth and associated pain. As such, treatment also includes a situation in which the pathological condition, or at least one symptom characterized by the pathological condition, is completely suppressed, for example, prevented from occurring, or stopped, for example, terminated, so that the host no longer suffers from the pathological condition or at least one symptom characterized by the pathological condition.

[0302] Various hosts (the term “host” is used interchangeably with the terms “subject,” “individual,” and “patient”) can be treated according to the target method. Generally, these hosts are “mammals” or “mammals,” and these terms are widely used to describe organisms within the class Mammalia, including the order Carnivora (e.g., dogs and cats), the order Rodentia (e.g., mice, guinea pigs, and rats), and the order Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host will be a human.

[0303] For example, a kit containing a unit dose of the target antibody within an oral or injectable dosage is provided. In some embodiments, in addition to the container containing the unit dose, there may be an information package insert describing the use of the antibody and the associated benefits in treating the pathological condition of interest.

[0304] nucleic acids

[0305] The present invention provides a nucleic acid comprising a nucleotide sequence encoding a target antibody. The nucleotide sequence encoding the target antibody may be operably linked to one or more regulatory elements, such as a promoter and an enhancer, that allow the expression of the nucleotide sequence in an intended target cell (e.g., a cell genetically modified to synthesize and / or secrete the encoded antibody).

[0306] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus ultra-early promoters; herpes simplex virus thymidine kinase promoters; early and late SV40 promoters; promoters present in the long terminal repeats of retroviruses; mouse metallothionein-I promoters; and various tissue-specific promoters known in the art, but are not limited thereto.

[0307] The nucleotide sequence encoding the target antibody may be present in the expression vector and / or cloning vector. If the target antibody contains two or more distinct polypeptides, the nucleotide sequences encoding the two polypeptides may be cloned in the same or distinct vectors. The distinct polypeptides may be expressed from a single nucleic acid or a single vector using various schemes, such as distinct promoters, one or more internal ribosome entry sites (IRES), one or more autocleavage sequences (e.g., 2A cleavage sequences, e.g., P2A, T2A, E2A, and F2A), combinations thereof, etc. The expression vector may include selectable markers, replication origins, and other features that provide replication and / or maintenance of the vector.

[0308] Many suitable vectors and promoters are known to those skilled in the art; many are commercially available to produce the target recombinant construct. The following vectors are provided as examples. Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0309] Expression vectors generally have a convenient restriction site located near the promoter sequence to provide for the insertion of a nucleic acid sequence encoding a heterogeneous protein. Selectable markers that operate in the expression host may be present. Suitable expression vectors are viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (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; see 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...., see PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., see Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., viral vectors based on rat leukemia virus, splenic necrosis virus, and Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus), and vectors derived from retroviruses such as mammary tumor virus); etc., but are not limited thereto.

[0310] As mentioned above, the target nucleic acid comprises a nucleotide sequence encoding the target multiple-specific antibody. The target nucleic acid may comprise a nucleotide sequence encoding heavy and light chain CDRs, including MDR1 CDR and CD47 CDR. In some embodiments, the target nucleic acid comprises a nucleotide sequence encoding the heavy and / or light chain MDR1 CDR, and the CDR-coding sequence is interspersed with FR-coding nucleotide sequences. In some embodiments, the target nucleic acid comprises a nucleotide sequence encoding the heavy and / or light chain CD47 CDR, and the CDR-coding sequence is interspersed with FR-coding nucleotide sequences. In some embodiments, the FR-coding nucleotide sequence is a human FR-coding nucleotide sequence.

[0311] In some embodiments, the target nucleic acid comprises a nucleotide sequence encoding an amino acid sequence 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% sequence identity with respect to the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0312] In some embodiments, the target nucleic acid comprises a nucleotide sequence encoding an amino acid sequence 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% sequence identity with respect to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0313] In some embodiments, the target nucleic acid comprises a nucleotide sequence encoding an amino acid sequence 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% sequence identity with respect to the following amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK.

[0314] Nucleic acids may be introduced into cells, for example, by bringing the cells into contact with the nucleic acids, as described herein. Cells having the introduced nucleic acids will generally be referred to herein as genetically modified cells. Various nucleic acid delivery methods may be used, including, but not limited to, naked nucleic acid delivery, viral delivery, chemical transfection, biometrics, etc.

[0315] cell

[0316] The present invention provides isolated genetically modified cells (e.g., in vitro cells, ex vivo cells, cultured cells, etc.) that have been genetically modified with a target nucleic acid. In some embodiments, the target isolated genetically modified cells may produce a target antibody. In some cases, the genetically modified cells may deliver the antibody to a required target, for example. In some cases, the genetically modified cells may be used for the production, screening, and / or discovery of multispecific antibodies. The genetically modified cells may also, in some cases, comprise cells in which endogenous gene expression is reduced, e.g., suppressed, knocked down, or abolished, e.g., knocked out. The genetically modified cells may also, in some cases, comprise cells in which gene expression is enhanced, e.g., increased expression of endogenous genes or increased expression of heterologous genes.

[0317] Suitable cells include eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., bacterial cells. The introduction of the target nucleic acid into the host cell may be influenced, for example, by calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0318] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 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. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc.

[0319] In some cases, the useful mammalian cells may include cells derived from mammalian tissues or organs. In some cases, the cells used are kidney cells, and include, for example, kidney cells of an established kidney cell line, such as HEK 293T cells.

[0320] Suitable yeast cells, fungal cells, or algal cells are, but are not limited to, Peachia Pastoris ( Pichia pastoris ), Piccia Finlandica( Pichia finlandica ), Peachia trehalophila( Pichia trehalophila ), Peachia Coquelame( Pichia koclamae ), Peachia membrane nafaciens( Pichia membranaefaciens ), Peachia Opuntiae( Pichia opuntiae ), Peachia thermophilus ( Pichia thermotolerans ), Peachia Salictaria( Pichia salictaria ), Peachia Guercum ( Pichia guercuum ), Peachia Fizzperi( Pichia pijperi ), Peachia stifftis( Pichia stiptis ), Peachia methaneolica( Pichia methanolica ), Peachia species( Pichia sp. ), Saccharomyces cerevisiae( Saccharomyces cerevisiae ), Saccharomyces species ( Saccharomyces sp. ), Hansenula Polymorpha( Hansenula polymorpha ), Cluiveromyces species( Kluyveromyces sp. ), Cluveveromyces lactis( Kluyveromyces lactis ), Candida albicans( Candida albicans ), Aspergillus nidulans( Aspergillus nidulans ), Aspergillus niger( Aspergillus niger ), Aspergillus oryzae( Aspergillus oryzae ), Trichoderma reisei( Trichoderma reesei ), Chrysosporium lucnowense ( Chrysosporium lucknowense ), Fusarium species ( Fusarium sp. ), Fusarium gramineum( Fusarium gramineum ), Fusarium Venenatum( Fusarium venenatum ), Neurospora Crassa( Neurospora crassa ), Chlamydomonas reinharty ( Chlamydomonas reinhardtii Includes ), etc.

[0321] Suitable prokaryotic cells are, but are not limited to, E. coli ( Escherichia coli ), Lactobacillus species ( Lactobacillus sp.), Salmonella species ( Salmonella sp.), Shigella species ( Shigella It includes various laboratory strains such as sp.), etc. For example, Carrier et al. (1992) J. Immunol . 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science Refer to 270:299-302. Examples of Salmonella strains that can be used in the present invention include Salmonella typhi ( Salmonella typhi ) and Salmonella typhimurium ( S. typhimurium ...includes, but is not limited to. Suitable Shigella strains include Shigella flexneri ( Shigella flexneri ), Sigela Sone ( Shigella sonnei), and Shigella discenteriae ( Shigella disenteriae Includes, but is not limited to. Typically, laboratory strains are non-pathogenic. Other non-limiting examples of suitable bacteria are Bacillus subtilis ( Bacillus subtilis ), Pseudomonas pudita( Pseudomonas pudita ), Pseudomonas aeruginosa( Pseudomonas aeruginosa ), Pseudomonas mevaloni( Pseudomonas mevalonii ), Rhodobacter Sparrowides( Rhodobacter sphaeroides ), Rhodobacter capsulelatus ( Rhodobacter capsulatus ), Rhodospirilum rubrum( Rhodospirillum rubrum ), Rhodococcus species ( Rhodococcus Includes, but is not limited to, sp.), etc. In some embodiments, the host cell is Escherichia coli.

[0322] In some cases, the cells of the present invention may be immune cells. As used herein, the term “immune cell” generally includes leukocytes (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. “Immune cell” includes, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). “T cell” includes all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg), and gamma-delta T cells. “Cytotoxic cell” includes CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses.

[0323] In some cases, useful cells expressing the multispecific antibodies of the present invention may include producer T cells. Non-limiting examples of producer T cells include those described in Tsai & Davila Oncoimmunology. (2016) 5(5): e1122158 (the full text of which is incorporated herein by reference). Producer T cells engineered to include a nucleic acid sequence encoding the multispecific antibodies of the present invention may, in some cases, be used to deliver the antibodies to a target in need.

[0324] The cells of the present invention also include cells genetically modified to alter 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 the analysis of binding of multispecific antibodies, including those produced according to the description and methods provided herein, but not limited to those produced in accordance with the description and methods provided herein. In some cases, MDR1 may be knocked out or knocked down in the target cell line. In some cases, CD47 may be knocked out or knocked down in the target cell line. In some cases, MDR1 may be constitutively or inducibly overexpressed in the target cell line. In some cases, CD47 may be constitutively or inducibly overexpressed in the target cell line. In some cases, both MDR1 and CD47 may be knocked down, knocked out, or constitutively or inducibly overexpressed in the target cell line. Any convenient and appropriate method may be used for knockdown, knockout, and / or overexpression. The introduced nucleic acid may be stably integrated or exist transiently.

[0325] In some embodiments, the cells of the present invention comprise a genetically modified human cell line comprising an exogenous nucleic acid containing a sequence encoding MDR1 for overexpression of MDR1 and expressing CD47. In these cells, CD47 expression may be derived endogenously or exogenously (i.e., introduced), and MDR1 expression may be stable or transient. In some cases, the cell line of the present invention expressing CD47 may be configured to produce a genetically modified human cell that expresses CD47 and stably overexpresses MDR1.

[0326] The cells and cell lines of the present invention may be cultured, for example, by using the culture methods described herein. In some cases, cells into which nucleic acids have been introduced to genetically modify the cells may be cultured to produce cell lines. Useful cell lines may include, for example, genetically modified cell lines expressing CD47 and stably overexpressing MDR1, including human cell lines, but are not limited thereto.

[0327] The cells of the present invention and their cell lines may be used in various methods of the present invention, for example, as test samples, controls, etc. For example, in some cases, cells in which MDR1 and / or CD47 are knocked out and / or knocked down may be used as reference cells, for example, to which the binding of the multispecific antibody of the present invention can be compared. Other useful reference cells include, for example, non-cancerous cells, as well as normal cells and cells expressing various proteins at normal levels, such as normal levels of MDR1 and / or CD47, but are not limited thereto.

[0328] method

[0329] As summarized above, the method of the present invention comprises a method of contacting a cell with an antibody of the present invention, a method of treating a subject according to a method comprising the step of administering the antibody of the present invention to a subject, and a method of preparing the elements described in the present application, e.g., a multispecific antibody, a composition and preparation, a nucleic acid, an expression vector, a cell, etc.

[0330] As summarized above, the method of the present invention comprises the step of contacting cancer cells with the multispecific antibody of the present invention to promote and / or enhance the killing of cancer cells, for example. In some cases, the killing of cancer cells is mediated by an immune response or immune cells acting on the cancer cells as a result of opsonization of the cancer cells by bispecific targeting when two targets are co-expressed on the cancer cells. In some cases, the killing of cancer cells is mediated by an immune response or immune cells acting on the cancer cells as a result of masking or antagonism of CD47 epitopes present on the surface of the cancer cells by the multispecific antibody. In some cases, the killing of cancer cells is mediated by the inhibition of cell efflux of the cancer cells as a result of MDR1 antagonism on the cancer cells by the multispecific antibody. In some cases, the cells contacted with the multispecific antibody may be multidrug-resistant cancer cells. A method comprising the step of contacting cancer cells with the multispecific antibody of the present invention may or may not include the step of contacting the cancer cells with an additional therapy or active agent, such as, for example, chemotherapy agents, immunotherapy, radiation therapy, etc.

[0331] The step of contacting cancer cells with the multispecific antibody of the present invention will generally enhance the killing of cancer cells compared to, for example, the level of killing of cancer cells in the absence of the multispecific antibody. In some cases, when an additional activator is used, enhanced killing of cancer cells can be observed compared to the level of killing observed when using the additional activator alone. The amount of enhancement in cancer cell killing attributable to the multispecific antibody will vary and may range from a 5% increase in cancer cell killing to at least 90%, but is not limited to, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, etc. Such an increase may be compared to contact with one or more additional activators alone.

[0332] 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 labeling (e.g., using one or more cell viability stains), etc.

[0333] Treatment methods

[0334] The present invention provides a method for treating cancer, the method generally comprising the step of administering an effective amount of a target multispecific antibody to an individual (e.g., an individual with cancer) who requires the antibody alone (e.g., as monotherapy) or in combination with one or more additional therapeutic agents (e.g., as combination therapy). The administration of the multispecific antibody of the present invention may be carried out by any convenient and suitable delivery route.

[0335] An aspect of the present invention comprises a bispecific antibody molecule according to the preceding section of the specification for use in a method for treating cancer in a subject, the method comprising the step of administering the antibody to the subject. The method comprises the step of administering the antibody in combination with at least one additional activator, the at least one additional activator comprising a chemotherapy agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof. In a specific aspect, the at least one additional activator is a chemotherapy agent, and optionally the chemotherapy agent is taxol, a vinca alkaloid, or an anthracycline. Some chemotherapy agents that are substrates for the MDR1 pump include paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide, and nilotinib.

[0336] A chemotherapy agent for use in a method of treating cancer in a subject is also disclosed herein, the method comprising the step of administering the chemotherapy agent to the subject in an antibody combination disclosed herein, optionally the chemotherapy agent is taxol, vinca alkaloid, or an anthracycline.

[0337] Accordingly, administration includes, but is not limited to,, for example, antibody delivery by injection, antibody delivery by infusion, delivery of a nucleic acid or expression vector encoding a multispecific antibody, and delivery of an antibody by administration of a cell expressing and secreting a multispecific antibody to a target. Administration of an agent, a nucleic acid encoding an agent, a cell expressing an agent, etc. may include contact with the agent, contact with the nucleic acid, contact with the cell, etc.

[0338] In some embodiments, the effective amount of the target multispecific antibody is an amount effective for reducing 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, in one or more doses compared to the severity of adverse symptoms when not treated with the antibody.

[0339] In some embodiments, the effective amount of the target multispecific antibody is an amount effective for improving cancer in a treated subject (i.e., slowing down cancer growth, stopping cancer growth, reversing cancer growth, and killing cancer cells (e.g., tumor cells, etc.) in one or more doses when administered alone (e.g., as monotherapy) or in combination with one or more additional therapeutic agents (e.g., as combination therapy). For example, the effective amount of the target antibody can reduce the rate of cancer growth or reduce the size of the 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%, or more compared to when there is no treatment with the multispecific antibody in the subject.

[0340] In some cases, the subject may be treated systemically, for example, with a target multispecific antibody, with or without one or more additional reagents. "Systemic treatment" means treatment that is not limited to targeting a specific tumor (e.g., a primary tumor or a limited secondary tumor) or a specific cancer-containing tissue (e.g., the liver in the case of liver cancer, blood in the case of blood cancer, etc.), as used herein. Systemic treatment generally applies to the subject's entire body and, but is not limited to, may include, for example, total body radiation therapy, total body chemotherapy, total body immunotherapy, combinations thereof, etc.

[0341] In some cases, the subject may be treated locally, for example, with a target multispecific antibody, with or without one or more additional reagents. "Local treatment" means a treatment that is specifically related to the location of the tumor (e.g., primary tumor or limited secondary tumor) or specifically related to the cancer-containing tissue (e.g., liver in the case of liver cancer, blood in the case of blood cancer, etc.). In some cases, local treatment may also be administered in a manner to affect the environment surrounding the tumor, such as the tissue surrounding the tumor or the tissue immediately adjacent to the tumor. Local treatment will generally not affect or be targeted to tissues located away from the site of cancer, including the tumor, such as the site of the primary tumor. Useful local treatments that may be administered in addition to or in combination with the target multispecific antibody include, but are not limited to, surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof.

[0342] In some embodiments, the target treatment method comprises the step of administering the target multispecific antibody and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, chemotherapy agents, radiotherapy agents, immunotherapy agents, other antibodies or multispecific antibody agonists, etc. Additional therapies that may be administered to the subject before, during, or after the target step of administering the multispecific antibody of the present invention will depend on many factors, including, for example, the type of cancer, the subject's medical history, general health condition and / or any comorbidities, etc. Useful cancer therapies include, but are not limited to, radiotherapy, chemotherapy, immunotherapy, etc.

[0343] Radiation therapy includes, but is not limited to, X-rays or gamma rays delivered from an externally applied source, such as a beam, or by the implantation of a small radioactive source.

[0344] Antibodies suitable for use in cancer treatment include naked antibodies, e.g., trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), olegovomab (OvaRex™), lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), etc., and conjugated antibodies, e.g., gemtuzumab ozogamicin (Mylortarg™), brentuximab vedotin (Adcetris™), 90Y-labeled ibritumomab tiuxetane (Zevalin™), 131I-labeled tocitumomab (Bexxar™), etc., are included but not limited thereto. Antibodies suitable for use in cancer treatment also include but are not limited to antibodies that have occurred against tumor-associated antigens. These antigens include but are not limited to CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Ley, VEGF, VEGFR, integrin alpha-V-beta-3, integrin alpha-5-beta-1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, Tenacine, etc.

[0345] Conventional cancer therapies are also, for example, but are not limited to, ado-trastuzumab emtansine (Kadcyla) targeted HER2 (ERBB2 / neu) (approved for use in breast cancer); afatinib (Gilotrif) targeted EGFR (HER1 / ERBB1), HER2 (ERBB2 / neu) (approved for use in non-small cell lung cancer); aldesleukin (Proleukin) targeted (approved for use in renal cell carcinoma and melanoma); alectinib (Alecensa) targeted ALK (non-small cell lung cancer); alemtuzumab (Campath) targeted CD52 (approved for use in B-cell chronic lymphocytic leukemia); atezolizumab (Tecentriq) targeted PD-L1 (approved for use in urothelial carcinoma and non-small cell lung cancer); Avelumab (Bavencio) targeted PD-L1 (Merkel cell carcinoma); Axitinib (Inlyta) targeted kit, PDGFRβ, VEGFR1 / 2 / 3 (approved for use in renal cell carcinoma); Belimumab (Benlysta) targeted BAFF (approved for use in lupus erythematosus); Velinostat (Beleodaq) targeted HDAC (approved for use in peripheral T-cell lymphoma); Bevacizumab (Avastin) targeted VEGF ligand (approved for use in cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma, non-small cell lung cancer, ovarian cancer, peritoneal cancer, and renal cell carcinoma); Blinatumomab (Blincyto) targeted CD19 / CD3 (approved for use in acute lymphoblastic leukemia (precursor B-cell); Bortezomib (Velcade) targeted proteasome (approved for use in multiple myeloma and mantle cell lymphoma); Bosutinib (Bosulif) targeted ABL (approved for use in chronic myeloid leukemia);Brentuximab Vedotin (Adcetris) targets CD30 (approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma); Brigatinib (Alunbrig) targets ALK (approved for use in non-small cell lung cancer (ALK+); Cabozantinib (Cabometyx, Cometriq) targets FLT3, KIT, MET, RET, and VEGFR2 (approved for use in medullary thyroid carcinoma and renal cell carcinoma); Carfilzomib (Kyprolis) targets proteasomes (approved for use in multiple myeloma); Ceritinib (Zykadia) targets ALK (approved for use in non-small cell lung cancer); Cetuximab (Erbitux) targets EGFR (HER1 / ERBB1) (approved for use in colorectal cancer and squamous cell carcinoma of the head and neck); Cobimetinib (Cotellic) targeted MEK (approved for use in melanoma); Crizotinib (Xalkori) targeted ALK, MET, ROS1 (approved for use in non-small cell lung cancer); Dabrafenib (Tafinlar) targeted BRAF (approved for use in melanoma and non-small cell lung cancer); Daratumumab (Darzalex) targeted CD38 (approved for use in multiple myeloma); Dasatinib (Sprycel) targeted ABL (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); Denosumab (Xgeva) targeted RANKL (approved for use in giant cell tumors of bone); Dinutuximab (Unituxin) targeted B4GALNT1 (GD2) (approved for use in pediatric neuroblastoma); Durvalumab (Imfinzi) targeted PD-L1 (approved for use in urothelial carcinoma); Elotuzumab (Empliciti) targeted SLAMF7 (CS1 / CD319 / CRACC) (approved for use in multiple myeloma);Enasidenib (Idhifa) targeted IDH2 (approved for use in acute myeloid leukemia); Erlotinib (Tarceva) targeted EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer and pancreatic cancer); Everolimus (Afinitor) targeted mTOR (approved for use in neuroendocrine tumors of pancreatic, gastrointestinal, or pulmonary origin, renal cell carcinoma, non-resected subependymal giant cell astrocytoma, and breast cancer); Gefitinib (Iressa) targeted EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer); Ibritumomab tiuxetane (Zevalin) targeted CD20 (approved for use in non-Hodgkin's lymphoma); Ibrutinib (Imbruvica) targeted BTK (approved for use in mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenstrom's macroglobulinemia); Idelalisib (Zydelig) targeted PI3Kδ (approved for use in chronic lymphocytic leukemia, follicular B-cell non-Hodgkin lymphoma, and small lymphocytic lymphoma); Imatinib (Gleevec) targeted KIT, PDGFR, and ABL (approved for use in GI stromal tumors (KIT+), dermatofibrosarcoma protuberans, and multiple hematological malignancies); Ipilimumab (Yervoy) targeted CTLA-4 (approved for use in melanoma); Ixazomib (Ninlaro) targeted proteasome (approved for use in multiple myeloma); Ripatinib (Tykerb) targets HER2 (ERBB2 / neu) and EGFR (HER1 / ERBB1) (approved for use in breast cancer (HER2+); Lenvatinib (Lenvima) targets VEGFR2 (approved for use in renal cell carcinoma and thyroid cancer); Midostaurin (Rydapt) targets FLT3 (approved for use in acute myeloid leukemia (FLT3+);Necitumumab (Portrazza) targeted EGFR (HER1 / ERBB1) (approved for use in squamous and non-small cell lung cancer); Neratinib (Nerlynx) targeted HER2 (ERBB2 / neu) (approved for use in breast cancer); Nilotinib (Tasigna) targeted ABL (approved for use in chronic myelodysplastic leukemia); Niraparib (Zejula) targeted PARP (approved for use in ovarian cancer, fallopian tube cancer, and peritoneal cancer); Nivolumab (Opdivo) targeted PD-1 (approved for use in colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, melanoma, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma); Obinutuzumab (Gazyva) targeted CD20 (approved for use in chronic lymphocytic leukemia and follicular lymphoma); Ofatumumab (Arzerra, HuMax-CD20) targeted CD20 (approved for use in chronic lymphocytic leukemia); Olaparib (Lynparza) targeted PARP (approved for use in ovarian cancer); Olaratumab (Lartruvo) targeted PDGFRα (approved for use in soft tissue sarcoma); Osimertinib (Tagrisso) targeted EGFR (approved for use in non-small cell lung cancer); Palbociclib (Ibrance) targeted CDK4, CDK6 (approved for use in breast cancer); Panitumumab (Vectibix) targeted EGFR (HER1 / ERBB1) (approved for use in colorectal cancer); Panobinostat (Farydak) targeted HDAC (approved for use in multiple myeloma); Pazopanib (Votrient) targeted VEGFR, PDGFR, KIT (approved for use in renal cell carcinoma); Pembrolizumab (Keytruda) targeted PD-1 (approved for use in classic Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+), head and neck squamous cell carcinoma, solid tumors (MSI-H);Pertuzumab (Perjeta) targets HER2 (ERBB2 / neu) (approved for use in breast cancer (HER2+); Ponatinib (Iclusig) targets ABL, FGFR1-3, FLT3, VEGFR2 (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); Ramucirumab (Cyramza) targets VEGFR2 (approved for use in colorectal cancer, gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, and non-small cell lung cancer); Regorafenib (Stivarga) targets KIT, PDGFRβ, RAF, RET, VEGFR1 / 2 / 3 (approved for use in colorectal cancer, gastrointestinal stromal tumors, and hepatocellular carcinoma); Ribociclib (Kisqali) targets CDK4 and CDK6 (approved for use in breast cancer (HR+, HER2-); Rituximab (Rituxan, Mabthera) targeted CD20 (approved for use in non-Hodgkin lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis); Rituximab / hyaluronidase human (Rituxan Hycela) targeted CD20 (approved for use in chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma); Romidepsin (Istodax) targeted HDAC (approved for use in cutaneous T-cell lymphoma and peripheral T-cell lymphoma); Rucaparib (Rubraca) targeted PARP (approved for use in ovarian cancer); Ruxolitinib (Jakafi) targeted JAK1 / 2 (approved for use in myelofibrosis); Siltuximab (Sylvant) targeted IL-6 (approved for use in multicentric Castleman's disease); cipulucel-T (Provenge) targeted (approved for use in prostate cancer);Sondezib (Odomzo) targeted Smoothened (approved for use in basal cell carcinoma); Sorafenib (Nexavar) targeted VEGFR, PDGFR, KIT, RAF (approved for use in hepatocellular carcinoma, renal cell carcinoma, and thyroid carcinoma); Temsirolimus (Torisel) targeted mTOR (approved for use in renal cell carcinoma); Tocitumomab (Bexxar) targeted CD20 (approved for use in non-Hodgkin lymphoma); Trametinib (Mekinist) targeted MEK (approved for use in melanoma and non-small cell lung cancer); Trastuzumab (Herceptin) targeted HER2 (ERBB2 / neu) (approved for use in breast cancer (HER2+) and gastric cancer (HER2+); Includes targeted therapies for cancer, including vandetanib (Caprelsa) targeting EGFR (HER1 / ERBB1), RET, and VEGFR2 (approved for use in medullary thyroid carcinoma); vemurafenib (Zelboraf) targeting BRAF (approved for use in melanoma); venetoclax (Venclexta) targeting BCL2 (approved for use in chronic lymphocytic leukemia); bismodezip (Erivedge) targeting PTCH and Smoothend (approved for use in basal cell carcinoma); vorinostat (Zolinza) targeting HDAC (approved for use in cutaneous T-cell lymphoma); zib-aflibercept (Zaltrap) targeting PIGF and VEGFA / B (approved for use in colorectal cancer); etc.;

[0346] Biological reaction modifiers suitable for use with the method of the present invention include, but are not limited to: (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) interferon-α; (7) interferon-γ; (8) colony-stimulating factors; (9) angiogenesis inhibitors; and (10) antagonists of tumor necrosis factors.

[0347] Chemotherapy agents are non-peptide (i.e., non-protein) compounds that reduce the proliferation of cancer cells, and include cytotoxic agents and cell arrest agents. Non-limiting examples of chemotherapy agents include alkylating agents, nitrosoureas, metabolic antagonists, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0348] Agents that act to reduce cell proliferation are known in the art and are widely used. These agents include, but are not limited to, alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazenes, and include but are limited to mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysine), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozosin, chlorozotosin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipovroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0349] Metabolic antagonist agents include, but are not limited to, folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), fluxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazofolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0350] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindecin, etc.; podophyllotoxins, e.g., etoposide, teniposide, etc.; antibiotics, e.g., anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, ceruvidin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives, etc.; phenoxyzone biscyclopeptides, e.g., dactinomycin; Basic glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mitramycin); anthracenediones, e.g., mitoxantrone; azirinopyrrolindolediones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin, etc.; etc., but are not limited thereto.

[0351] Other antiproliferative cytotoxic agents are Nabelben, CPT-11, anastrozol, letrazol, capecitabine, leroxapine, cyclophosphamide, ifosamide, and droloxapine.

[0352] Agents that affect microtubules and have antiproliferative activity are also suitable for use, but are not limited to allocolchicine (NSC 406042), halicondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), meitansine (NSC 153858), lyzoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, natural and synthetic epotillons, but are not limited to epotillon A, epotillon B, discodermolid; estramustine, nocodazole, etc.

[0353] Hormone modulators and steroids (e.g., synthetic analogs) suitable for use include adrenocorticosteroids, e.g., prednisone, dexamethasone, etc.; estrogens and progestins, e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticosteroids, e.g., aminoglutethimide; 17α-ethinylestradiol; Diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), tremiphene (Fareston), and zoladex are included, but not limited thereto. Estrogens stimulate proliferation and differentiation, and therefore compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0354] Other chemotherapy agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidophilotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other antiproliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, deoxysperguarin, azasporine, leflunomide, mizoribin, azaspiran (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazolin); etc.

[0355] "Taxane" includes paclitaxel as well as any active taxane derivative or prodrug. "Paclitaxel" (to be understood herein to include analogs, formulations, and derivatives, e.g., docetaxel, Taxol™, TAXOTERE™ (formulations of docetaxel), 10-desacetyl analogs of paclitaxel and 3'N-desbenzoyl-3'Nt-butoxycarbonyl analogs of paclitaxel) can be readily manufactured using techniques known to those skilled in the art (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; See 5,229,529; and EP 590,267), or various commercial suppliers, e.g., Sigma Chemical Co., St. Louis, Mo. (taxus brevifolia( Taxus brevifolia T7402 of ) or Taxus yananensis ( Taxus yannanensis It can be obtained from the T-1912 of ).

[0356] It should be understood that paclitaxel refers not only to the common chemically available form of paclitaxel, but also to analogs and derivatives (e.g., Taxotere™, docetaxel, as mentioned above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, or paclitaxel-albumin).

[0357] Various known derivatives, including both hydrophilic and hydrophobic derivatives, are included within the term "taxane." Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivatives described in U.S. Patent No. 5,415,869. It further includes, but is not limited to, prodrugs of paclitaxel, including those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0358] Useful immunotherapies include: anti-PD-1 / PD-L1 immunotherapy, and / or other immunotherapeutic targets that may be targeted in the treatment method, e.g., immune checkpoint markers, e.g., CTLA-4, LAG-3, and TIM-3. Anti-PD-1 / PD-L1 immunotherapy comprises, but is not limited to, the step of administering an effective amount of one or more anti-PD-1 / PD-L1 therapeutic antagonists to a subject, and such antagonists include, but are not limited to, OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), Tecentriq™ (atezolizumab), durvalumab (MEDI4736), durvalumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242, etc.

[0359] CTLA-4, also known as CD152, binds to CD80 and CD86. Antibodies against CTLA-4 have been approved for treating certain types of cancer. The co-inhibitory effect of CTLA-4 with other immunotherapies makes it a promising candidate for use in combination with other immunotherapies to treat specific cancers. TIM-3 can also be targeted for immunotherapy against various cancer types.

[0360] LAG-3 is currently undergoing clinical trials for cancer treatment. Anti-LAG-3 immunotherapy involves the use of antagonist LAG-3 antibodies capable of inhibiting induced (i.e., antigen-specific) Treg inhibitory activity by activating T effector cells (by downregulating LAG-3 inhibitory signals to pre-activated LAG-3+ cells). Useful LAG-3 antagonist antibodies include relatlimab (BMS-986016; developed by Bristol-Myers Squibb), IMP701 (developed by Imutep), TSR-033 (anti-LAG-3 mAb; developed by TESARO, Inc.), etc.

[0361] Immunotherapy also includes T cell-based immunotherapy, e.g., adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T cell therapy. For example, a subject may be administered a population of CAR T cells engineered to target antigens expressed by the subject's cancer. T cell-based therapy may, in some cases, involve obtaining cell samples, such as blood samples or tumor biopsies, from the subject, with or without genetic modification of the cultured immune cells, and culturing immune cells from the samples in vitro. For example, immune cells may be obtained from the subject, cultured in vitro, and modified into CARs specific to antigens expressed by the cancer to produce a population of CAR T cells. The CAR T cells may then be reintroduced into the subject to target the cancer. T cell-based immunotherapy may be constructed in various ways depending on the specific cancer being treated, for example, by targeting various antigens, by collecting / culturing various cell types, etc. In addition, T cell-based immunotherapy can be administered systemically, for example, by intravenous injection, or locally, for example, by infusion (e.g., intraperitoneal injection, pleural catheter injection, etc.), direct injection, etc.

[0362] In some cases, the treatment methods described herein may include, but are not limited to, the step of administering to the subject one or more inhibitors of a multidrug resistance transporter, including, for example, a multidrug resistance transporter other than MDR1. Useful inhibitors of a multidrug resistance transporter may include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and small molecule inhibitors. Inhibitors of a multidrug resistance transporter include ABC transporter inhibitors. A summary of such MDR modulators or reverters is provided in Choi (2005), Cancer Cell Int, 5:30 (the full text of which is incorporated herein by reference).

[0363] Individuals suitable for treatment using the method of the present invention include: an individual having cancer; an individual diagnosed with cancer; an individual being treated for cancer by chemotherapy, radiation therapy, antibody therapy, surgery, etc.; an individual who has been treated for cancer (e.g., one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) and has failed to respond to the treatment; and an individual who has been treated for cancer (e.g., one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) and initially responded to the treatment but later recurred, i.e., the cancer has recurred.

[0364] The method of the present invention can be used to target and treat various cancers, including, for example, primary cancer, secondary cancer, regrowth cancer, recurrent cancer, refractory cancer, etc. For example, in some cases, the method of the present invention can be used as an initial treatment for primary cancer identified in a subject. In some cases, the method of the present invention can be used as a non-primary (e.g., secondary or subsequent) treatment in, for example, a subject with cancer that is refractory to prior treatment, a subject with cancer that regrows after prior treatment, a subject showing a mixed response to prior treatment (e.g., a positive response for at least one tumor in the subject and a negative or neutral response for at least a second tumor in the subject), etc.

[0365] In some cases, the method of the present invention may be used to treat subjects with drug-resistant cancer, such as multidrug-resistant cancer. Multidrug resistance (MDR) is a mechanism in which many cancers develop resistance to chemotherapy drugs, causing minimal cell death and expansion of drug-resistant tumors. MDR cancers may involve one or more resistance mechanisms, including, but not limited to, increased expression of efflux pumps, reduced uptake of drugs, inhibition of cell death or apoptosis, regulation of drug metabolism, etc. In some cases, the method of the present invention may prevent, reverse, or avoid MDR.

[0366] In some cases, the method of the present invention may include the step of treating a subject with cancer resistant to the first agent with an effective amount of the target multispecific antibody described herein in combination with a second agent different from the first agent. For example, in some cases, the cancer of the subject may be resistant to the first chemotherapy agent, and the subject may be treated by administering an effective amount of the target multispecific antibody described herein in combination with a second chemotherapy agent different from the first chemotherapy agent. Various combinations of the first chemotherapy agent and the second chemotherapy agent may be used, for example, depending on the type of cancer being treated, the likelihood of resistance developing, etc.

[0367] Many cancers are known to develop drug resistance. For these and other reasons, the method of the present invention is, but is not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma, etc.), anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma (extrahepatic), bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma and malignant fibrous histiocytoma, etc.), brainstem glioma, brain tumor (e.g., astrocytoma, central nervous system germ cell tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma), e.g.), breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumors, Burkitt lymphoma, carcinoid tumors (e.g., pediatric, gastrointestinal, etc.), carcinoma of unknown primary lesion, cardiac (heart) tumors, central nervous system (e.g., atypical organomorphic / rod-shaped tumors, embryonic tumors, germ cell tumors, lymphoma, etc.), cervical cancer, pediatric cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelodysplastic leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, duct (e.g., bile duct, extrahepatic, etc.), tubular carcinoma in situ (DCIS), embryonic tumors, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma, Ewing sarcoma, extracranial germ cell tumors, extratesticular germ cell tumors, extrahepatic cholangiocarcinoma, ocular cancer (e.g., ocular Melanoma, retinoblastoma, etc.),Fibrous Histiocytoma of Bone (e.g., malignant, osteosarcoma, etc.), gallbladder cancer, gastric cancer (gastric cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (e.g., extracranial, extratesticular, ovarian, testicular, etc.), Gestational Trophoblastic Disease, glioma, morphogenetic leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), histiocytosis (e.g., Langerhans cell, etc.), Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (e.g., pancreatic endocrine tumor, etc.), Kaposi sarcoma, renal 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 (AML), Chronic Lymphocytic (CLL), Chronic Myelodysplastic (CML), Cryogenic, etc.), lip and oral cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), lymphoma (e.g., AIDS-associated, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system (CNS), etc.), macroglobulinemia (e.g., Waldenstrom, etc.), male breast cancer, bone malignant fibrous histiocytoma and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell cervical cancer with latent primary, intermediate carcinoma with NUT gene, oral cancer, Multiple Endocrine Neoplasia Syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, Myelodysplastic Syndrome, myelodysplastic / spinal proliferative neoplasm, myelodysplastic leukemia (e.g., chronic (CML), etc.), myeloid leukemia (e.g., acute (AML), etc.),Spinal proliferative neoplasms (e.g., chronic, etc.), nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer (e.g., lip, etc.), oropharyngeal cancer, osteosarcoma and osteomalignant fibrous histiocytoma, ovarian cancer (e.g., epithelial, germ cell tumor, low-malignancy potential tumor, etc.), pancreatic cancer, pancreatic endocrine tumor (islet cell tumor), papillomatosis, paraganglioma, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), ureteral, transitional cell carcinoma, retinoblastoma, It can be used to treat various cancers including rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue, uterine, etc.), Sezary Syndrome, skin cancer (e.g., pediatric, melanoma, Merkel cell carcinoma, non-melanoma, etc.), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer (e.g., latent, primary, metastatic, etc.), gastric cancer (gastric cancer), T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, ureteropelvic transitional cell carcinoma, urethral cancer, urethral cancer, uterine cancer (e.g., endometrial, etc.), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor, etc.

[0368] The treatment methods described herein may, in some cases, be performed on subjects who have previously undergone one or more conventional treatments. For example, in the case of oncology, the methods described herein may, in some cases, be performed after conventional cancer therapy, including, but not limited to, conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc.

[0369] In some cases, the method described herein may be used when the subject does not respond to or is refractory to conventional therapy. In other cases, the method described herein may be used when the subject has responded to conventional therapy.

[0370] In some cases, the method of the present invention may be used to target, treat, or eliminate a subject with minimal residual disease (MRD) remaining after prior cancer therapy. Targeting, treatment, and / or clearance of MRD may be pursued using the method regardless of whether it is determined to be refractory or intractable to prior treatment. In some cases, the method of the present invention may be used to target, treat, or eliminate a subject with MRD after determining that the MRD is refractory to prior treatment or one or more available treatment options other than using the multispecific antibodies described herein.

[0371] In some cases, this method may be used prophylactically for surveillance. For example, when a subject in need has no detectable disease but is at risk of developing recurrent cancer, such as drug-resistant cancer, a treatment involving one or more of the multispecific antibodies described herein may be administered. In some cases, a prophylactic approach may be used when the subject is at particularly high risk of developing primary cancer that is predicted to be drug-resistant or expected to become drug-resistant. In some cases, a prophylactic approach may be used when the subject has previously been treated for cancer and is at risk of recurrence or development of drug resistance.

[0372] In some cases, the method of the present invention may involve the step of analyzing cancer for the expression of one or more markers or therapeutic targets. For example, in some cases, the method may involve the step of analyzing a cancer sample of 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.

[0373] In some cases, whether a subject is treated with the multispecific antibody of the present invention may depend on the results of TAA and / or MDR1 tests. For example, in some cases, if the cancer expresses TAA above a predetermined threshold, the subject may be treated with the multispecific antibody of the present invention, and if the cancer expresses TAA below a predetermined threshold, the subject may not be treated with the multispecific antibody, and, for example, the subject may be treated with conventional therapy for the appropriate cancer without the target multispecific antibody. In some cases, if the cancer expresses MDR1 above a predetermined threshold, the subject may be treated with the multispecific antibody of the present invention, and if the cancer expresses MDR1 below a predetermined threshold, the subject may not be treated with the multispecific antibody, and, for example, the subject may be treated with conventional therapy for the appropriate cancer without the target multispecific antibody. In some cases, if the cancer expresses both TAA and MDR1 above a predetermined threshold, the subject may be treated with the multispecific antibody of the present invention, and if the cancer expresses TAA and MDR1 below a predetermined threshold, the subject may not be treated with the multispecific antibody, for example, the subject may be treated with a conventional regimen for the appropriate cancer without the target multispecific antibody.

[0374] To analyze MDR1 and / or TAA levels, any convenient analysis may be used, including but not limited to, flow cytometry, nucleic acid-based analysis (e.g., amplification, sequencing, etc.), cytometric analysis, immunohistochemistry, etc. Any convenient biological sample may be used, including but not limited to, for example, cancer biopsy samples. A predetermined threshold useful for evaluating the expression of one or more markers and / or targets may be determined by any convenient and appropriate method, including comparison of the measured expression levels to a corresponding control. For example, in some cases, a predetermined threshold useful for the levels of MDR1 and / or TAA analyzed in a sample may correspond to the levels of MDR1 and / or TAA as measured in reference cells, e.g., healthy / normal cells. TAA may be CD47, PD-L1, or EGFR.

[0375] manufacturing method

[0376] As summarized above, the method of the present invention also includes a method for preparing and / or identifying multispecific antibodies as described herein. The target antibody may be produced by any known method, e.g., protein synthesis; recombinant DNA methods; and other conventional synthesis methods.

[0377] If the target antibody is a single-strand polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When polypeptides are chemically synthesized, the synthesis can proceed through a liquid or solid phase. Solid-phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by the sequential addition of the remaining amino acids, is an example of a suitable method for the chemical synthesis of target antibodies. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing target antibodies. Techniques for solid-phase synthesis are referenced in 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. It is described in 12:723-8. Briefly, small insoluble, porous beads are treated as functional units to form peptide chains. After repeated cycles of coupling and deprotection, the attached free N-terminal amine on the solid phase is coupled to a single N-protected amino acid unit. This unit is deprotected to reveal a new N-terminal amine to which additional amino acids can be attached. The peptide remains immobilized on the solid phase and undergoes a filtration process before being cleaved.

[0378] Standard recombinant methods may be used for the production of target antibodies. For example, nucleic acids encoding light chain and heavy chain variable regions that are selectively linked to constant regions are inserted into an expression vector. The light chain and heavy chain may be cloned from the same or different expression vectors. A DNA fragment encoding the immunoglobulin chain is operably linked to a control sequence of the expression vector(s) that ensures the expression of the immunoglobulin polypeptide. The expression control sequence includes, but is not limited to, a promoter (e.g., naturally associated or heterogeneous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. The expression control sequence may be a eukaryotic promoter system within the vector capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for the expression of high levels of nucleotide sequences and the collection and purification of antibodies.

[0379] Due to the degeneration of the code, various nucleic acid sequences can encode the respective immunoglobulin amino acid sequence. The desired nucleic acid sequence can be produced by de novo solid-phase DNA synthesis of an early-manufactured variant of the desired polynucleotide or by polymerase chain reaction (PCR) mutagenesis. Oligonucleotide-mediated mutagenesis is an example of a suitable method for producing 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 incorporate the oligonucleotide primer and synthesize the entire second complementary strand of the template encoding the selected change in the target polypeptide DNA.

[0380] Suitable expression vectors are typically replicated in a host organism as components of episomal or host chromosomal DNA. Commonly, expression vectors contain a selection marker (e.g., ampicillin-resistant, hygromycin-resistant, tetracycline-resistant, kanamycin-resistant, or neomycin-resistant) to allow detection of the cells transformed with the desired DNA sequence.

[0381] Escherichia coli is an example of a prokaryotic host cell that can be used to encode a target antibody-encoding polynucleotide. Other microbial hosts suitable for use include Bacillus, such as Bacillus subtilis, and other enteric bacteria, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors containing expression control sequences (e.g., replication origin) that are typically compatible with the host cell can also be prepared. In addition, any various widely known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda, will be present. The promoter will typically have ribosome binding site sequences, etc., to selectively control expression with operator sequences and to initiate and complete transcription and translation.

[0382] Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., Saccharomyces cerevisiae) and Pichia are examples of suitable yeast host cells, and suitable vectors have expression control sequences (e.g., promoters), replication origins, termination sequences, etc., if desired. Typical promoters include 3-phosphoglycerate kinase and other sugar-degrading enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

[0383] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell cultures) may also be used to express and produce polypeptides of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B-cells or hybridomas. Expression vectors for these cells may include expression control sequences, e.g., replication origin, promoter, and enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and essential information processing sites, e.g., ribosome binding site, RNA splicing site, polyadenylation site, and transcription terminator sequence. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0384] Once synthesized (chemically or by recombination), the whole antibody, its dimer, individual light and heavy chains, or other forms of the target antibody (e.g., scFv, etc.) may be purified according to standard procedures in the field, including ammonium sulfate precipitation, affinity column, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (generally see Scopes, Protein Purification (Springer-Verlag, NY, (1982))). The target antibody may be substantially pure, for example, at least about 80% to 85%, at least about 85% to 90%, at least about 90% to 95%, or 98% to 99%, or higher, and there are no contaminants such as cell debris, macromolecules, etc., other than, for example, the target antibody, etc.

[0385] In some embodiments, the method for generating a multispecific antibody of the present invention may include the step of producing candidate antibodies and screening them for activity. This method may generate a multispecific antibody that specifically binds to cells expressing both MDR1 and CD47 through the use of a series of steps. The steps of this method may include: producing a multispecific antibody or a plurality of antibodies that each contain or are expected to contain 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 MDR1 or CD47 with the multispecific antibody or the plurality of antibodies; comparing the binding of the multispecific antibody to the second cell with the binding of the multispecific antibody or the plurality of antibodies to the first cell to determine a binding specificity ratio; and identifying the multispecific antibody, or one or more of the plurality of antibodies, as specific to cells expressing both MDR1 and CD47 when the ratio is higher than a predetermined threshold. When such thresholds are used for the combination of comparisons, the thresholds may vary and may be in the range of 1.5:1 or higher, and include, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1, etc., but are not limited thereto.

[0386] Various cells may be used in this method, and, but are not limited to, include, for example, the cells described herein. In some cases, binding of the antibody to both MDR1-only expressing cells and CD47-only expressing cells may be performed. For example, in some cases, the method may include a case where, in connection with the step described above, the second cell expresses MDR1 rather than CD47, and the method further includes the step of contacting a third cell that does not express MDR1 but expresses CD47 with a multispecific antibody.

[0387] In some cases, this method may utilize one or more controls, including, but is not limited to, control cells, control reagents, etc. Useful control cells include those exhibiting known expression or a deficiency of known expression of one or more appropriate genes or proteins. Useful control reagents may include, but are not limited to, control antibodies, for example, monospecific antibodies against known targets. For example, in some cases, this method of the present invention may further include the step of contacting a first cell, a second cell, and / or a third cell with a control antibody selected from monospecific anti-MDR1 antibodies and monospecific anti-CD47 antibodies. Depending on the specific method used, various other or additional controls may be utilized where appropriate.

[0388] Kit

[0389] Aspects of the present invention also include a kit. The kit may include, for example, any combination of the multispecific antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. described herein. The target kit may include one or more of the following: a target multispecific antibody, a nucleic acid encoding it, or a cell containing the target multispecific nucleic acid. The kit may be configured for various purposes, for example, a therapeutic kit (for example, the kit may include a multispecific antibody and, for example, one or more additional activators, e.g., chemotherapy agents), a kit for antibody production, a kit for antibody screening, etc.

[0390] The optional components of the kit will vary and may include, for example, buffers; protease inhibitors; etc. If the target kit contains a target nucleic acid, the nucleic acid may also have restriction sites, multiple cloning sites, primer sites, etc. The various components of the kit may exist in separate containers, or certain compatible components may be pre-assembled into a single container if desired.

[0391] In addition to the components mentioned above, the target kit may include instructions for using the components of the kit to carry out the target method. Instructions for carrying out the target method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic, etc. As such, the instructions may be present in the kit as packaging inserts in the labeling of the kit's container or its components (i.e., related to the packaging or subpackaging). In other embodiments, the instructions exist as electronic storage data on a suitable computer-readable storage medium, for example, a compact disc-read-only memory (CD-ROM), a digital multi-purpose disc (DVD), a diskette, etc. In other embodiments, the actual instructions do not exist within the kit, but means for obtaining the instructions from a remote source, for example, via the Internet, are provided. An example of this embodiment is a kit containing a web address on which the instructions can be viewed and / or downloaded. Like the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0392] Non-limiting aspects of the exemplary invention

[0393] The aspects of the subject matter described above, including specific examples, may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the description mentioned above, specific non-limiting aspects of the invention are provided below. As will be apparent to those skilled in the art when reading the invention, each numbered aspect may be used or combined with any preceding or subsequent individual numbered aspects. This is intended to provide support for any combination of such 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 may be made without departing from the spirit or scope of the invention.

[0394] 1. A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and tumor-associated antigen (TAA), wherein the antibody molecule comprises two identical variable light chains (VL), a first variable heavy chain (VH), and a second VH chain, and

[0395] 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 VL chains, and

[0396] Bispecific antibodies bind to cancer cells expressing both MDR1 and TAA but exhibit reduced binding to non-cancer cells expressing MDR1 and / or TAA.

[0397] 2. As a bispecific antibody molecule of Embodiment 1, the antigen-binding sites of the two VL chains comprise light chain CDR 1-3 (LCDR 1-3) of the VL chain having the following sequence:

[0398] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (Sequence No.:1) (where X 1 is N, Q, or S).

[0399] 3. As a bispecific antibody molecule of Embodiment 2, the two VL chains comprise LCDR 1-3 of the VL chains having the following sequences:

[0400] (i) DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (Sequence No.: 2);

[0401] (ii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID No.: 3); or

[0402] (iii) DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (Sequence No: 4).

[0403] 4. As a bispecific antibody molecule of embodiment 2 or 3,

[0404] (i) LCDR1 is the following sequence: RSSQSIVHSTGX 1 Includes TYLE (sequence number: 5);

[0405] (ii) LCDR2 contains the following sequence: KISNRFS (sequence number: 6);

[0406] (iii) LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7).

[0407] (Here X 1 is N, Q, or S).

[0408] 5. As a bispecific antibody molecule of any one of embodiments 1 to 4, two VL chains are humanized.

[0409] 6. A bispecific antibody molecule of any one of embodiments 2 to 5, wherein two VL chains comprise the following sequence or amino acids that are at least 90% identical to the following sequence:

[0410] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO:8);

[0411] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO:3); or

[0412] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK (SEQ ID NO:4).

[0413] 7. As a bispecific antibody molecule of Embodiment 6, the two VL chains comprise the following sequence or a sequence that is at least 90% identical to the following sequence:

[0414] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO:8).

[0415] 8. A bispecific antibody molecule of any one of embodiments 1 to 7, wherein the antigen-binding site of the first VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence:

[0416] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence No.: 9) (where X 2 is N, Q, or S).

[0417] 9. A bispecific antibody molecule of any one of embodiments 1 to 8, wherein the antigen-binding site of the first VH chain comprises HCDR 1-3 of the VH chain having the following sequence:

[0418] (i) EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:10), or

[0419] (ii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence No.: 11), or

[0420] (iii) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:12).

[0421] 10. As a bispecific antibody molecule of embodiment 8 or 9,

[0422] (i) HCDR1 contains the following sequence: RYTMS (sequence number: 13);

[0423] (ii) HCDR2 is the following sequence: TISSGGG X 2 Contains TYYPDSVKG (sequence number: 14);

[0424] (iii) HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15).

[0425] (Here X 2 is N, Q, or S).

[0426] 11. As a bispecific antibody molecule of any one of embodiments 1 to 10, the first and / or second VH chain is humanized.

[0427] 12. As a bispecific antibody molecule of any one of embodiments 8 to 11, 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:

[0428] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGnTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:16);

[0429] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:11); or

[0430] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:12).

[0431] 13. As a bispecific antibody molecule of any one of embodiments 2 to 12,

[0432] (i) LDR1 contains the following sequence: RSSQSIVHSTGNTYLE (sequence number: 26), and

[0433] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0434] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0435] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0436] HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG (sequence number: 29), and

[0437] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0438] (ii) LCDR1 contains the following sequence: RSSQSIVHSTGQTYLE (sequence number: 27), and

[0439] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0440] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0441] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0442] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG (sequence number: 30), and

[0443] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0444] (iii) LCDR1 contains the following sequence: RSSQSIVHSTGSTYLE (sequence number: 28), and

[0445] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0446] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0447] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0448] HCDR2 contains the following sequence: TISSGGGSTYYPDSVKG (sequence number: 31), and

[0449] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0450] (iv) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE (sequence number: 26), and

[0451] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0452] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0453] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0454] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG (sequence number: 30), and

[0455] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0456] (v) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE (sequence number: 26), and

[0457] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0458] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0459] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0460] HCDR2 contains the following sequence: TISSGGGSTYYPDSVKG (sequence number: 31), and

[0461] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0462] (vi) LCDR1 contains the following sequence: RSSQSIVHSTGQTYLE (sequence number: 27), and

[0463] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0464] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0465] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0466] HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG (sequence number: 29), and

[0467] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0468] (vii) LCDR1 contains the following sequence: RSSQSIVHSTGSTYLE (sequence number: 28), and

[0469] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0470] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0471] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0472] HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG (sequence number: 29), and

[0473] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0474] (viii) LCDR1 contains the following sequence: RSSQSIVHSTGSTYLE (sequence number: 28), and

[0475] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0476] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0477] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0478] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG (sequence number: 30), and

[0479] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15), or

[0480] (ix) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE (sequence number: 26), and

[0481] LCDR2 contains the following sequence: KISNRFS (sequence number: 6), and

[0482] LCDR3 contains the following sequence: FQASHFPRT (sequence number: 7);

[0483] HCDR1 contains the following sequence: RYTMS (sequence number: 13), and

[0484] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG (sequence number: 30), and

[0485] HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15).

[0486] 14. As a bispecific antibody molecule of any one of embodiments 1 to 7, the second VH chain is derived from a monospecific antibody molecule that binds to TAA, and when paired with one of the light chains, the affinity of the bispecific antibody molecule for TAA is at least twice lower than the affinity of the monospecific antibody molecule for TAA from which the VH chain is derived.

[0487] 15. As a bispecific antibody molecule of any one of embodiments 1 to 7, TAA is CD47.

[0488] 16. As a bispecific antibody molecule of embodiment 15, the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH sequence comprising the following amino acid sequence:

[0489] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:17).

[0490] 17. As a bispecific antibody molecule of embodiment 15 or 16, the second VH chain comprises HCDR1: NYNMH (sequence number: 18) comprising the following sequence, HCDR2: TIYPGNDDTSYNQKFKD (sequence number: 19) comprising the following sequence, and HCDR3: GGYRAMDY (sequence number: 20) comprising the following sequence.

[0491] 18. As a bispecific antibody molecule of embodiment 15 or 16, the second VH chain comprises the following amino acid sequence or an amino acid that is at least 90% identical to the following amino acid sequence:

[0492] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:17),

[0493] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:21),

[0494] EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO:22), or

[0495] QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO:23).

[0496] 19. As a bispecific antibody molecule of any one of embodiments 1 to 14, TAA is PD-L1.

[0497] 20. As a bispecific antibody molecule of Embodiment 19, the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0498] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32).

[0499] 21. As a bispecific antibody molecule of embodiment 19 or 20, the antigen-binding site of the second VH chain comprises HCDR1: DSWIH (SEQ No.: 33) comprising the following sequence, HCDR2: WISPYGGSTYYADSVKG (SEQ No.: 34) comprising the following sequence, and HCDR3: RHWPGGFDY (SEQ No.: 35) comprising the following sequence.

[0500] 22. As a bispecific antibody molecule of any one of embodiments 19 to 21, the second VH chain comprises the following amino acid sequence or an amino acid that is at least 90% identical to the following amino acid sequence:

[0501] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32).

[0502] 23. As a bispecific antibody molecule of any one of embodiments 1-14, TAA is EGFR.

[0503] 24. As a bispecific antibody molecule of Embodiment 23, the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0504] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO:36).

[0505] 25. As a bispecific antibody molecule of embodiment 23 or 24, the antigen-binding site of the second VH chain comprises HCDR1: SGDYYWS (SEQ No.: 37), HCDR2: YIYYSGSTDYNPSLKS (SEQ No.: 38), and HCDR3: VSIFGVGTFDY (SEQ No.: 39).

[0506] 26. As a bispecific antibody molecule of embodiment 24 or 25, 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:

[0507] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO:36).

[0508] 27. As a bispecific antibody molecule of Embodiment 23, the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0509] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:40).

[0510] 28. As a bispecific antibody molecule of embodiment 26 or 27, the second VH chain comprises HCDR1: NYGVH (sequence number: 41) comprising the following sequence, HCDR2: VIWSGGNTDYNTPFTS (sequence number: 42) comprising the following sequence, and HCDR3: ALTYYDYEFAY (sequence number: 43) comprising the following sequence.

[0511] 29. As a bispecific antibody molecule of any one of embodiments 26 to 28, 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:

[0512] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:40).

[0513] 30. As a bispecific antibody molecule of Embodiment 1, the antigen-binding sites of the two VL chains comprise light chain CDR 1-3 (LCDR 1-3) of the VL chain having the following sequence:

[0514] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25).

[0515] 31. As a bispecific antibody molecule of Embodiment 30,

[0516] (i) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE (sequence number: 44);

[0517] (ii) LCDR2 contains the following sequence: KISRLEA (sequence number: 45);

[0518] (iii) LCDR3 contains the following sequence: FQGSHFPRT (sequence number: 46).

[0519] 32. As a bispecific antibody molecule of embodiment 30 or 31, 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:

[0520] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25).

[0521] 33. As a bispecific antibody molecule of any one of embodiments 30-32, the antigen-binding site of the first VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence:

[0522] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA.

[0523] 34. As a bispecific antibody molecule of Embodiment 33,

[0524] (i) HCDR1 contains the following sequence: SYTMS;

[0525] (ii) HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG;

[0526] (iii) HCDR3 contains the following sequence: YYRYEAWFAS.

[0527] 35. As a bispecific antibody molecule of any one of embodiments 30-34, 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:

[0528] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA.

[0529] 36. As a bispecific antibody molecule of any one of embodiments 30-32, the antigen-binding site of the first VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence:

[0530] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence No.: 9) (where X 2 is N, Q, or S).

[0531] 37. As a bispecific antibody molecule of Embodiment 36,

[0532] (i) HCDR1 contains the following sequence: RYTMS (sequence number: 13);

[0533] (ii) HCDR2 contains the following sequence: TISSGGG X2TYYPDSVKG (sequence number: 14);

[0534] (iii) HCDR3 contains the following sequence: YGAGDAWFAY (sequence number: 15).

[0535] (Here X 2 is N, Q, or S).

[0536] 38. As a bispecific antibody molecule of embodiments 36-37, 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:

[0537] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:16);

[0538] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:11); or

[0539] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO:12).

[0540] 39. As a bispecific antibody molecule of any one of embodiments 1-7, the antigen-binding site of the first VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence:

[0541] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA.

[0542] 40. As a bispecific antibody molecule of Embodiment 39,

[0543] (i) HCDR1 contains the following sequence: SYTMS;

[0544] (ii) HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG;

[0545] (iii) HCDR3 contains the following sequence: YYRYEAWFAS.

[0546] 41. As a bispecific antibody molecule of embodiments 39-40, the first VH chain comprises the following sequence or amino acids that are at least 90% identical to the following sequence:

[0547] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA.

[0548] 42. As a bispecific antibody molecule of any one of embodiments 30-41, the second VH chain is derived from a monospecific antibody molecule that binds to TAA, and the affinity of the bispecific antibody molecule for TAA is at least twice lower than the affinity of the monospecific antibody molecule for TAA from which the VH chain is derived when paired with one of the light chains.

[0549] 43. As a bispecific antibody molecule of any one of embodiments 30-42, TAA is CD47.

[0550] 44. As a bispecific antibody molecule of Embodiment 43, the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0551] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:17).

[0552] 45. As a bispecific antibody molecule of embodiment 44, the second VH chain comprises HCDR1: NYNMH (sequence number: 18) comprising the following sequence, HCDR2: TIYPGNDDTSYNQKFKD (sequence number: 19) comprising the following sequence, and HCDR3: GGYRAMDY (sequence number: 20) comprising the following sequence.

[0553] 46. ​​As a bispecific antibody molecule of embodiment 44 or 45, the second VH chain comprises the following amino acid sequence or an amino acid sequence that is at least 90% identical to the following sequence:

[0554] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:17),

[0555] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO:21),

[0556] EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO:22), or

[0557] QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO:23).

[0558] 47. As a bispecific antibody molecule of any one of embodiments 30-42, TAA is PD-L1.

[0559] 48. As a bispecific antibody molecule of Embodiment 47, the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0560] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32).

[0561] 49. As a bispecific antibody molecule of embodiment 47 or 48, the second VH chain comprises HCDR1: DSWIH (sequence number: 33) comprising the following sequence, HCDR2: WISPYGGSTYYADSVKG (sequence number: 34) comprising the following sequence, and HCDR3: RHWPGGFDY (sequence number: 35) comprising the following sequence.

[0562] 50. As a bispecific antibody molecule of embodiment 48 or 49, 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:

[0563] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32).

[0564] 51. As a bispecific antibody molecule of any one of embodiments 30-42, TAA is EGFR.

[0565] 52. As a bispecific antibody molecule of embodiment 51, the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0566] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO:36).

[0567] 53. As a bispecific antibody molecule of embodiment 52, the second VH chain comprises HCDR1: SGDYYWS (sequence number: 37) comprising the following sequence, HCDR2: YIYYSGSTDYNPSLKS (sequence number: 38) comprising the following sequence, and HCDR3: VSIFGVGTFDY (sequence number: 39) comprising the following sequence.

[0568] 54. As a bispecific antibody molecule of embodiment 52 or 53, 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:

[0569] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO:36).

[0570] 55. As a bispecific antibody molecule of embodiment 51, the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain comprising the following amino acid sequence:

[0571] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:40).

[0572] 56. As a bispecific antibody molecule of embodiment 52, the antigen binding site of the second VH chain comprises HCDR1: NYGVH (sequence number: 41) comprising the following sequence, HCDR2: VIWSGGNTDYNTPFTS (sequence number: 42) comprising the following sequence, and HCDR3: ALTYYDYEFAY (sequence number: 43) comprising the following sequence.

[0573] 57. As a bispecific antibody molecule of embodiment 52 or 53, 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:

[0574] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:40).

[0575] 58. As a bispecific antibody molecule of any one of embodiments 1 to 57, the antibody has a 2x greater affinity for cells expressing both MDR1 and TAA compared to cells expressing MDR1 or TAA.

[0576] 59. As a bispecific antibody molecule of any one of embodiments 1 to 58, the antibody can increase the sensitivity of cancer cells to treatment with a chemotherapy agent, and the half-maximum inhibitory concentration (IC50) of the chemotherapy agent is a VH chain having the following sequence when co-administered with the antibody:

[0577] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO:24); and

[0578] VL chain with the following sequence:

[0579] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25)

[0580] When co-administered with the anti-MDR1 antibody 15D3 containing, the IC50 is at least 2 times lower than that of the chemotherapy drug, and optionally, the IC50 is measured in vitro.

[0581] 60. As a bispecific antibody molecule of embodiment 59, the cancer cells are NALM6 ADR cells and selectively the chemotherapy agent includes paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide, or nilotinib.

[0582] 61. As a bispecific antibody molecule of any one of embodiments 1 to 60, the antibody inhibits efflux by MDR1 when it binds to a cell expressing MDR1.

[0583] 62. As a bispecific antibody molecule of any one of embodiments 1 to 1, the antibody has a VH chain having the following sequence:

[0584] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA (SEQ ID NO:24); and

[0585] VL chain with the following sequence:

[0586] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25)

[0587] The anti-MDR1 antibody containing 15D3 binds to MDR1 with an affinity at least twice lower than that of 15D3.

[0588] 63. A bispecific antibody molecule of any one of embodiments 1 to 62, wherein the antibody comprises an Fc domain modified to reduce or abolish the binding of the antibody to one or more Fcγ receptors.

[0589] 64. As a bispecific antibody molecule of any one of embodiments 1 to 63 for use in a method for treating cancer in a subject, the method comprises the step of administering the antibody to the subject.

[0590] 65. As a bispecific antibody molecule for use according to embodiment 64, the method comprises the step of administering the antibody in combination with at least one additional activator, wherein the at least one additional activator comprises a chemotherapy agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof.

[0591] 66. As a bispecific antibody molecule for use according to embodiment 65, at least one additional activator is a chemotherapy agent, optionally the chemotherapy agent is taxol, vinca alkaloid, or anthracycline.

[0592] 67. A chemotherapy agent to be used in a method for treating cancer in a subject, the method comprises the step of administering the chemotherapy agent in combination with an antibody of any one of embodiments 1 to 63, optionally the chemotherapy agent is taxol, vinca alkaloid, or anthracycline.

[0593] 68. A bispecific antibody molecule for use according to modality 67, wherein the subject being treated has cancer determined to be resistant to chemotherapy drugs.

[0594] 69. A method for treating a subject for cancer, wherein the method comprises the step of administering to the subject a therapeutically effective amount of a bispecific antibody molecule of any one of embodiments 1 to 63.

[0595] 70. As a method of embodiment 69, the method comprises the step of administering a bispecific antibody molecule in combination with at least one additional activator, wherein the at least one additional activator comprises a chemotherapy agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof.

[0596] 71. As a method of embodiment 70, at least one additional active agent is a chemotherapy agent, optionally the chemotherapy agent is taxol, vinca alkaloid, or anthracycline.

[0597] 72. As a method of embodiment 71, the subject to be treated has cancer determined to be resistant to treatment with a chemotherapy agent, and optionally the chemotherapy agent comprises paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide, or nilotinib.

[0598] 73. Variable light chain (VL) containing a light chain CDR (LCDR) of the VL chain having the following sequence:

[0599] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (here X 1 is N, Q, or S); and

[0600] Variable heavy chain (VH) containing a heavy chain CDR (HCDR) of the VH chain with the following sequence:

[0601] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (where 2 is N, Q, or S)

[0602] Antibody containing; or

[0603] Variable light chain (VL) containing a light chain CDR (LCDR) of the VL chain with the following sequence:

[0604] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25); and

[0605] Variable heavy chain (VH) containing a heavy chain CDR (HCDR) of the VH chain with the following sequence:

[0606] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA

[0607] Antibody containing

[0608] 74. As an antibody of modality 73,

[0609] The VL chain contains LCDR 1-3 of the VL chain with the following sequences:

[0610] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK;

[0611] The VH chain contains HCDR 1-3 of the VH chain with the following sequences:

[0612] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or

[0613] The VL chain contains LCDR 1-3 of the VL chain with the following sequences:

[0614] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGQTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK;

[0615] The VH chain contains HCDR 1-3 of the VH chain with the following sequences:

[0616] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS, or

[0617] The VL chain contains LCDR 1-3 of the VL chain with the following sequences:

[0618] DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGSTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGQGTKLEIK;

[0619] The VH chain contains LCDR 1-3 of the VH chain with the following sequences:

[0620] EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS.

[0621] 75. As an antibody of modality 74,

[0622] LCDR1 includes the following sequence: RSSQSIVHSTGNTYLE, RSSQSIVHSTGQTYLE, or RSSQSIVHSTGSTYLE, and

[0623] LCDR2 contains the following sequence: KISNRFS,

[0624] LCDR3 contains the following sequence: FQASHFPRT.

[0625] 76. As an antibody of modality 75,

[0626] (i) HCDR1 contains the following sequence: RYTMS;

[0627] (ii) HCDR2 is the following sequence: TISSGGG X 2 Includes TYYPDSVKG;

[0628] (iii) HCDR3 contains the following sequence: YGAGDAWFAY

[0629] (Here X 2 is N, Q, or S).

[0630] 77. As an antibody of modalities 74-75,

[0631] (i) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE, and

[0632] LCDR2 contains the following sequence: KISNRFS,

[0633] LCDR3 contains the following sequence: FQASHFPRT;

[0634] HCDR1 contains the following sequence: RYTMS, and

[0635] HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG, and

[0636] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0637] (ii) LCDR1 contains the following sequence: RSSQSIVHSTGQTYLE, and

[0638] LCDR2 contains the following sequence: KISNRFS, and

[0639] LCDR3 contains the following sequence: FQASHFPRT;

[0640] HCDR1 contains the following sequence: RYTMS, and

[0641] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG, and

[0642] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0643] (iii) LCDR1 contains the following sequence: RSSQSIVHSTGSTYLE, and

[0644] LCDR2 contains the following sequence: KISNRFS, and

[0645] LCDR3 contains the following sequence: FQASHFPRT;

[0646] HCDR1 contains the following sequence: RYTMS, and

[0647] HCDR2 contains the following sequence: TISSGGGSTYYPDSVKG, and

[0648] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0649] (iv) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE, and

[0650] LCDR2 contains the following sequence: KISNRFS, and

[0651] LCDR3 contains the following sequence: FQASHFPRT;

[0652] HCDR1 contains the following sequence: RYTMS, and

[0653] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG, and

[0654] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0655] (v) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE, and

[0656] LCDR2 contains the following sequence: KISNRFS, and

[0657] LCDR3 contains the following sequence: FQASHFPRT;

[0658] HCDR1 contains the following sequence: RYTMS, and

[0659] HCDR2 contains the following sequence: TISSGGGSTYYPDSVKG, and

[0660] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0661] (vi) LCDR1 contains the following sequence: RSSQSIVHSTGQTYLE, and

[0662] LCDR2 contains the following sequence: KISNRFS, and

[0663] LCDR3 contains the following sequence: FQASHFPRT;

[0664] HCDR1 contains the following sequence: RYTMS, and

[0665] HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG, and

[0666] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0667] (vii) LCDR1 contains the following sequence: RSSQSIVHSTGSTYLE, and

[0668] LCDR2 contains the following sequence: KISNRFS, and

[0669] LCDR3 contains the following sequence: FQASHFPRT;

[0670] HCDR1 contains the following sequence: RYTMS, and

[0671] HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG, and

[0672] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0673] (viii) LCDR1 contains the following sequence: RSSQSIVHSTGSTYLE, and

[0674] LCDR2 contains the following sequence: KISNRFS, and

[0675] LCDR3 contains the following sequence: FQASHFPRT;

[0676] HCDR1 contains the following sequence: RYTMS, and

[0677] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG, and

[0678] HCDR3 contains the following sequence: YGAGDAWFAY, or

[0679] (ix) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE, and

[0680] LCDR2 contains the following sequence: KISNRFS, and

[0681] LCDR3 contains the following sequence: FQASHFPRT;

[0682] HCDR1 contains the following sequence: RYTMS, and

[0683] HCDR2 contains the following sequence: TISSGGGQTYYPDSVKG, and

[0684] HCDR3 contains the following sequence: YGAGDAWFAY.

[0685] 78. As an antibody of any one of embodiments 73-77, the VL chain of the antibody comprises the following amino acid sequence or comprises an amino acid sequence that is at least 90% identical to the following amino acid sequence:

[0686] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK,

[0687] 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:

[0688] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS .

[0689] 79. As the antibody of modality 73, the antibody is

[0690] Variable light chain (VL) containing light chain CDR 1-3 (LCDR) of the VL chain with the following sequence:

[0691] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25); and

[0692] Variable heavy chain (VH) containing heavy chain CDR 1-3 (HCDR) of the VH chain with the following sequence:

[0693] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA

[0694] Includes

[0695] 80. As an antibody of modality 79,

[0696] (i) LCDR1 contains the following sequence: RSSQSIVHSTGNTYLE (sequence number: 44);

[0697] (ii) LCDR2 contains the following sequence: KISRLEA (sequence number: 45);

[0698] (iii) LCDR3 contains the following sequence: FQGSHFPRT (sequence number: 46);

[0699] (i) HCDR1 contains the following sequence: SYTMS;

[0700] (ii) HCDR2 contains the following sequence: TISSGGGNTYYPDSVKG;

[0701] (iii) HCDR3 contains the following sequence: YYRYEAWFAS.

[0702] 81. As an antibody of embodiment 79 or 80, the VL 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: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (Sequence 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.

[0703] 82. As an antibody of any one of embodiments 73-81, the antibody is a monospecific divalent antibody that specifically binds to MDR-1.

[0704] 83. As an antibody of any one of modalities 73-81, the antibody is a bispecific antibody containing a VL chain as a common antagonist.

[0705] 84. As an antibody of embodiment 83, the bispecific antibody comprises an MDR-1 binding domain and a tumor-associated antigen (TAA) binding domain, and the MDR-1 binding domain and the TAA binding domain each comprise LCDR 1-3 of the VL chain.

[0706] 85. As the antibody of Embodiment 84, the TAA is CD47 and the TAA binding domain comprises HCDR 1-3 of the VH chain having the following sequence:

[0707] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0708] 86. As the antibody of embodiment 85, the VH chain of the CD47 binding domain comprises HCDR1: NYNMH containing the following sequence, HCDR2: TIYPGNDDTSYNQKFKD containing the following sequence, and HCDR3: GGYRAMDY containing the following sequence.

[0709] 87. As the antibody of Embodiment 84, the TAA is PD-L1 and the PD-L1 binding domain comprises HCDR 1-3 of the VH chain having the following sequence:

[0710] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32).

[0711] 88. As the antibody of embodiment 87, the VH chain of the PD-L1 binding domain comprises HCDR1: DSWIH (sequence number: 33), HCDR2: WISPYGGSTYYADSVKG (sequence number: 34), and HCDR3: RHWPGGFDY (sequence number: 35).

[0712] 89. As the antibody of Embodiment 84, the TAA is EGFR and the EGFR binding domain comprises an HCDR of a VH chain having the following sequence:

[0713] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO:36).

[0714] 90. As an antibody of embodiment 89, the VH chain of the EGFR binding domain comprises HCDR1: SGDYYWS (sequence number: 37), HCDR2: YIYYSGSTDYNPSLKS (sequence number: 38), and HCDR3: VSIFGVGTFDY (sequence number: 39).

[0715] 91. As the antibody of Embodiment 84, the TAA is EGFR and the EGFR binding domain comprises an HCDR of a VH chain having the following sequence:

[0716] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:40).

[0717] 92. As an antibody of embodiment 91, the VH chain of the EGFR binding domain comprises HCDR1: NYGVH (sequence number: 41), HCDR2: VIWSGGNTDYNTPFTS (sequence number: 42), and HCDR3: ALTYYDYEFAY (sequence number: 43).

[0718] 93. Variable light chain (VL) containing a light chain CDR (LCDR) of the VL chain having the following sequence:

[0719] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (here X 1 is N, Q, or S); or

[0720] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO:25); and

[0721] Variable heavy chain (VH) containing a heavy chain CDR (HCDR) of the VH chain with the following sequence:

[0722] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS; or

[0723] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:32); or

[0724] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO:36); or

[0725] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO:40)

[0726] Antibody containing

[0727] 94. As the antibody of Modification 93, the VL chain is (i) the following sequence: RSSQSIVHSTGX 1 LCDR1 containing TYLE (Sequence No. 5); (ii) LCDR2 containing the following sequence: KISNRFS (Sequence No. 6); and (iii) LCDR3 containing the following sequence: FQASHFPRT (Sequence No. 7) (where X 1 It contains N, Q, or S) and the VH chain is

[0728] (i) HCDR1: NYNMH containing the following sequence, HCDR2: TIYPGNDDTSYNQKFKD containing the following sequence, and HCDR3: GGYRAMDY containing the following sequence; or

[0729] (ii) HCDR1: DSWIH (SEQ No.: 33), HCDR2: WISPYGGSTYYADSVKG (SEQ No.: 34), and HCDR3: RHWPGGFDY (SEQ No.: 35); or

[0730] (iii) HCDR1: SGDYYWS (Sequence No.: 37), HCDR2: YIYYSGSTDYNPSLKS (Sequence No.: 38), and HCDR3: VSIFGVGTFDY (Sequence No.: 39); or

[0731] (iv) HCDR1: NYGVH (Sequence No.: 41), HCDR2: VIWSGGNTDYNTPFTS (Sequence No.: 42), and HCDR3: ALTYYDYEFAY (Sequence No.: 43)

[0732] Includes

[0733] 95. As the antibody of Embodiment 93, the VL chain comprises (i) LCDR1: RSSQSIVHSTGNTYLE (SEQ No.: 44) containing the following sequence; (ii) LCDR2: KISRLEA (SEQ No.: 45) containing the following sequence; and (iii) LCDR3: FQGSHFPRT (SEQ No.: 46) containing the following sequence, and the VH chain

[0734] HCDR1 containing the following sequence: NYNMH, HCDR2 containing the following sequence: TIYPGNDDTSYNQKFKD, and HCDR3 containing the following sequence: GGYRAMDY; or

[0735] HCDR1: DSWIH containing the following sequence (Sequence No.: 33), HCDR2: WISPYGGSTYYADSVKG containing the following sequence (Sequence No.: 34), and HCDR3: RHWPGGFDY containing the following sequence (Sequence No.: 35); or

[0736] HCDR1 containing the following sequence: SGDYYWS (Sequence No.: 37), HCDR2 containing the following sequence: YIYYSGSTDYNPSLKS (Sequence No.: 38), and HCDR3 containing the following sequence: VSIFGVGTFDY (Sequence No.: 39); or

[0737] HCDR1 containing the following sequence: NYGVH (Sequence No.: 41), HCDR2 containing the following sequence: VIWSGGNTDYNTPFTS (Sequence No.: 42), and HCDR3 containing the following sequence: ALTYYDYEFAY (Sequence No.: 43)

[0738] Includes

[0739] 96. As an antibody of any one of modes 93-95, the antibody is a bispecific antibody.

[0740] 97. As an antibody of any one of modalities 1-96, the antibody is a humanized antibody or a chimeric antibody containing a human Fc domain.

[0741] 98. As an antibody of modality 97, it comprises an immunoglobulin G1 (IgG1) Fc domain.

[0742] 99. As an antibody of any one of embodiments 1-98, the first VH chain is fused to the first Fc domain and the second VH chain is fused to the second Fc domain.

[0743] 100. As an antibody of embodiment 99, the Fc domain comprises a modified CH3 domain that preferentially forms a heterodimer comprising a first VH chain and a second VH chain.

[0744] 101. As an antibody of embodiment 100, the first and second Fc domains are human immunoglobulin G1 (IgG1) Fc domains.

[0745] 102. An antibody of any one of embodiments 1 to 101; and

[0746] Pharmaceutically acceptable excipients

[0747] A pharmaceutical composition comprising

[0748] 103. A pharmaceutical composition of embodiment 102 further comprises at least one additional active agent.

[0749] 104. As a pharmaceutical composition of embodiment 103, at least one additional active agent comprises a chemotherapeutic agent.

[0750] 105. As a pharmaceutical composition of embodiment 104, the chemotherapeutic agent is taxol, vinca alkaloid, or anthracycline.

[0751] 106. As a pharmaceutical composition of any one of embodiments 103 to 105, at least one additional activator comprises an inhibitor of a multidrug resistance transporter.

[0752] 107. As a pharmaceutical composition of embodiment 103, at least one additional active agent comprises an immunotherapeutic agent.

[0753] 108. One or more nucleic acids comprising one or more sequences encoding an antibody of any one of embodiments 1 to 107.

[0754] 109. As one or more nucleic acids of modality 108, one or more sequences are operably linked to a promoter.

[0755] 110. One or more recombinant expression vectors comprising one or more nucleic acids of modality 108 or 109.

[0756] 111. Mammalian cells genetically modified with one or more recombinant expression vectors of modality 110.

[0757] 112. As a cell of modality 111, the cell is an immune cell.

[0758] 113. An antibody of any one of embodiments 1 to 63 and 73-101 or a nucleic acid encoding said antibody; and

[0759] At least one additional active agent

[0760] A kit including

[0761] 114. As a kit of embodiment 113, at least one additional active agent comprises a chemotherapy agent, an inhibitor of a multidrug-resistant transporter, an immunotherapy agent, or a combination thereof.

[0762] 115. A method for killing cancer cells, the method comprises the step of contacting cancer cells with an antibody of any one of embodiments 1 to 63 and 73-101.

[0763] 116. As a method of embodiment 115, the method further comprises the step of administering at least one additional activator.

[0764] 117. As a method of embodiment 116, at least one additional activator comprises a chemotherapy agent.

[0765] 118. As a method of embodiment 116 or 117, the method increases the killing of cancer cells by at least 5% compared to when in contact with at least one additional activator alone.

[0766] 119. In any one of embodiments 115 to 118, the cancer cells are drug-resistant cancer cells.

[0767] 120. A method for treating a subject for cancer, comprising the step of administering to the subject an antibody of any one of embodiments 1 to 63 and 73-101 or a pharmaceutical composition of any one of embodiments 102-107.

[0768] 121. As a method of modality 120, the subject was previously treated for cancer.

[0769] 122. As a method of modality 120 or 121, the cancer is drug-resistant or multidrug-resistant.

[0770] 123. As a method of modality 122, cancer is resistant to chemotherapy drugs.

[0771] 124. As a method of modality 122, cancer is resistant to immunotherapy agents.

[0772] 125. As a method of any one of embodiments 120 to 122, the cancer is resistant to an inhibitor of a multidrug-resistant transporter.

[0773] 126. Any one of embodiments 120 to 125 further comprises the step of administering at least one additional activator to a subject.

[0774] 127. As a method of embodiment 126, at least one additional activator comprises a chemotherapy agent.

[0775] 128. As a method of modality 127, the chemotherapy agent is taxol, vinca alkaloid, or anthracycline.

[0776] 129. As a method of any one of embodiments 126 to 128, at least one additional activator comprises an inhibitor of a multidrug-resistant transporter.

[0777] 130. As a method of any one of embodiments 126 to 128, at least one additional activator comprises an immunotherapeutic agent.

[0778] 131. As a method of any one of embodiments 126 to 128, the method increases the effect of at least one additional activator compared to treatment with at least one additional activator alone.

[0779] 132. As a method of embodiment 131, the increased effect includes at least a 5% increase in cancer cell killing.

[0780] 133. Any one of embodiments 120 to 132 further comprises the step of analyzing a sample of cancer to determine whether the cancer expresses MDR1 above a predetermined threshold, expresses tumor-associated antigen (TAA) above a predetermined threshold, or both, optionally the TAA includes CD47.

[0781] 134. As a method of embodiment 133, a predetermined threshold corresponds to the level of MDR1 and / or TAA expressed by a reference cell.

[0782] 135. As a method of embodiment 134, MDR1 and / or TAA were knocked out or knocked down in a reference cell.

[0783] 136. As a method of modality 134 or 135, the reference cell is a non-cancerous cell.

[0784] 137. As a method of embodiment 134, non-cancerous cells express MDR1 and / or TAA at normal levels.

[0785] 138. In any one of embodiments 120 to 137, if the cancer expresses MDR1 and TAA above a predetermined threshold, the subject is administered a multispecific antibody, and if the cancer expresses MDR1 or TAA below a predetermined threshold, the subject is treated with a conventional regimen without administering a multispecific antibody.

[0786] 139. A method for generating a multispecific antibody that specifically binds to cells expressing both multidrug resistance protein 1 (MDR1) and tumor-associated antigen (TAA), wherein the TAA selectively comprises CD47, PD-L1, or EGFR, and the method

[0787] A step of producing a multispecific antibody comprising an MDR1-binding domain and a TAA-binding domain;

[0788] A step of contacting a first cell expressing MDR1 and TAA with a multispecific antibody;

[0789] A step of contacting a second cell expressing MDR1 or TAA with a multispecific antibody;

[0790] A step of comparing the binding of a multispecific antibody to a first cell with the binding of a multispecific antibody to a second cell to determine the binding specificity ratio.

[0791] Step of confirming that the multispecific antibody is specific to cells expressing both MDR1 and TAA when the ratio is higher than a predetermined threshold.

[0792] Includes

[0793] 140. As a method of modality 139, the predetermined threshold is greater than 2:1.

[0794] 141. As a method of embodiment 139 or 140, the method further comprises the step of contacting a third cell that expresses TAA but does not express MDR1 with a multispecific antibody.

[0795] 142. As a method of embodiment 139 or 140, the method further comprises the step of contacting a first cell, the second cell, and / or a third cell with a control antibody selected from a monospecific anti-MDR1 antibody and a monospecific anti-TAA antibody.

[0796] 143. A genetically modified human cell line, wherein the cell line expresses a tumor-associated antigen (TAA) and contains exogenous nucleic acid containing a sequence encoding multidrug resistance protein 1 (MDR1) for the overexpression of MDR1, optionally the TAA contains leukocyte surface antigen CD47.

[0797] 144. As the cell line of modality 143, the cell line is an elongated cell line.

[0798] 145. As the cell line of Modification 144, the cell line is the HEK 293T cell line.

[0799] 146. A method for producing any one of embodiments 143 to 145 of a cell line, the method comprising the step of producing a genetically modified human cell that expresses TAA and stably overexpresses MDR1 by contacting a human cell expressing TAA with an exogenous nucleic acid under conditions sufficient to introduce the exogenous nucleic acid into the cell; and

[0800] A step of culturing genetically modified human cells under conditions sufficient to produce a genetically modified human cell line that expresses TAA and stably overexpresses MDR1.

[0801] Includes

[0802] The following examples are provided as examples, not limiting ones.

[0803] Examples

[0804] The following examples are presented to provide those skilled in the art with a complete disclosure and description of the methods for manufacturing and using the invention, and are not intended to limit the scope of what the inventor considers to be the invention, but are intended to indicate all or only experiments in which the following experiments were performed. Although efforts have been made to ensure accuracy with respect to the figures used (e.g., amounts, temperatures, etc.), some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near it.

[0805] Common methods in molecular and cellular biochemistry can be found 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 these are incorporated herein by reference). The reagents, cloning vectors, cells, and kits mentioned in or relating to this method are available from commercial distributors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., etc., as well as from repositories such as, for example, Addgene, Inc., American Type Culture Collection (ATCC), etc.

[0806] Example 1: Generation of a bispecific mAb that specifically binds to and resensitizes cells simultaneously expressing Pgp (MDR1) and CD47, rather than cells with reduced or absent protein expression.

[0807] This example demonstrates the development of a molecule based on an antibody format capable of effectively blocking efflux by specifically targeting antigens expressed by cancer cells and binding to the extracellular domain of EP in a selective manner. Blocking EP results in the resensitization and killing of cells that are resistant to or will become resistant to chemotherapy. In this example, a bispecific antibody molecule was constructed that blocks efflux by binding to the extracellular domain (ECD) of EP and targeting cancer immune checkpoint proteins. The application of the bispecific antibody described below selectively resensitizes cells to chemotherapy when EP is co-expressed with the targeted immune checkpoint proteins.

[0808] Materials and Methods

[0809] Cell line and cell viability experiments

[0810] Pgp-expressing HEK 293T, MCF-7, N6ADR, and SKNF7 cell lines were obtained from the American Type Culture Collection. N6 / ADR cells are also referred to as NALM6 / ADR cells. All cell lines derived from them were maintained in humidified incubators at 37°C and 5% CO2 in RPMI 1640 or DMEM supplemented with up to 10% fetal bovine serum (Sigma), non-essential amino acids, and 2 mmol / L L-glutamine (unless otherwise indicated). Cells were used as supplied, modified to overexpress Pgp (Ox), or induced to express Pgp or knocked down with lentivirus-mediated short hairpin RNA (KD), or induced to knock out the functional CD47 gene (KO) by CRISPR / Cas-mediated knockout technology as described (Cong, L. et al. (2013) Science339, 819-823). To determine the IC50 of vincristin and paclitaxel, cells were plate-cultured in normal growth medium and allowed to adhere overnight. Paclitaxel or vincristin (Sigma) was added at serial dilutions, and any regulator was added within the range of 0 to 500 μM / L. Cell viability was measured after 72 h using the Celltiter-Glo luminescence cell viability assay (Promega). The concentration of the drug (IC50) that results in a 50% inhibition of cell viability was calculated from a multivariable curve analysis (GraphPad Prism software, GraphPad Software, Inc.) and determined from at least two replicates. In most experiments performed, cell lines that did not show a 50% reduction in cell viability in response to drug and / or regulator treatment were considered by definition not to have reached an IC50 and were listed as exhibiting an IC50 of >1000 nmol / L for paclitaxel or the drug / regulator combination under study.

[0811] A recombinant cell line stably expressing the described monoclonal antibody (mAb) was also produced.

[0812] Recombinant DNA technology

[0813] DNA was manipulated using standard methods described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is available in Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242. The amino acids of the antibody chains were numbered and cited according to Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., (1991).

[0814] DNA Sequence Determination

[0815] The DNA sequence was determined by double-strand sequencing.

[0816] DNA and protein sequencing analysis and sequence data management

[0817] The Vector NTI (ThermoFisher) software package was used for sequence mapping, analysis, annotation, and examples.

[0818] Cell culture technology and antibody production

[0819] Standard cell culture techniques described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc. are used.

[0820] 293 & CHO cells were used for the transient production of mAbs, Fab'2, Fab, and bispecific mAbs. Different antibody constructs were expressed in Expi293 cells (A14527, ThermoFisher) using polymer-based co-transfection. Cells were grown in suspensions containing mammalian expression vectors according to the manufacturer's recommendations.

[0821] To prepare the bispecific construct, cells were transfected with the corresponding expression vector in a 1:1:4 ratio (heavy chain KK: heavy chain DD: light chain). A 1:2 ratio (heavy chain: light chain) was used for standard antibody expression.

[0822] Cells were harvested by centrifugation on day 6 after transfection. Specifically, 1 μg of total encoding DNA per 1 ml of transfected culture was diluted in Opti-MEM® medium (Life Technologies) and incubated with Expifectamine reagent (Life Technologies) in the same medium for 20 minutes. The mixture was then added to Expi293® cells growing in a suspension in Expi293® expression medium (Life Technologies) at 2.5 million cells / ml at 37°C with an overlay of 8% CO2 as known. After 6 days, the medium containing the antibody composition was harvested by centrifugation.

[0823] Reagents and cell lines used in the test: binding, efflux blockade, cell sensitization to chemotherapy agents

[0824] Human embryonic kidney (HEK) cell line HEK 293FT (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 g / mL streptomycin in 5% CO2 incubation at 37°C.

[0825] 293T cells were transiently transfected with human P glycoprotein-tagged ORF clones within the pLenti-C-Myc-DDK-P2A-Puro plasmid using the optimized PEIPro™ transfection protocol (Polyplus). DNA and JetPEI® were each diluted in culture medium prior to gentle mixing for approximately 10 minutes. This mixing led to the formation of a transfection complex, which was directly added to the cell culture. Effluent blockade was measured using the Multidrug Resistance Direct Dye Effluent Assay (Chemicon) according to the manufacturer's protocol.

[0826] Sources of target sequences, antibody sequences, and specific anti-target antibody sequences

[0827] In pLenti-C-Myc-DDK-P2A-Puro, human-tagged ORF clones of P glycoprotein (Pgp), also known as multidrug resistance protein 1 (MDR1), (gene ABCB1) (NM_000927) were obtained from the anti-CD47 antibody (CC2C6, Seiffert M, et al. (1999) Blood 94:3633) of Origene, Biolegend, and the anti-ABCB1 JSB-1 (MAB4120) of Millipore.

[0828] Generation of a stable ABCB1 overexpression (Ox) cell line

[0829] To characterize both binding and in vitro efficiency, a cell line stably overexpressing ABCB1 was developed. Adherent 293T naive cells obtained from the American Type Culture Collection (ATCC) were used.

[0830] As characterized by flow cytometry using a commercially available ABCB1 antibody (Biolegend, clone 4E3.16), this cell line expresses ABCB1 endogenously at low to moderate levels on the cell surface. 293T naive cells were transfected with ABCB1 using Polyplus PEIpro reagent. Three days after transfection, cells were selected using Hygromycin B solution (Millipore Sigma). 14 days after continuous Hygromycin B selection, 293T cells were evaluated for ABCB1 cell surface expression. To ensure that untransfected cells would not expand in future cultures, batch sorting of ABCB1-positive 293T cells using fluorescence-activated cell sorting (FACS) was performed using FACSAriaI (BD Biosciences). Batch-sorted 293T ABCB1-overexpressing cells were expanded, and ABCB1 overexpression was subsequently reconfirmed. ABCB1 overexpressing cells were generated from 293T-CD47 knockout cells generated as described herein using a similar method.

[0831] Generation of a stable ABCB1 KD 293T cell line

[0832] To characterize both defects and in vitro efficiency, a cell line with stably knocked ABCB1 expression was developed. Lentiviruses were produced in 293T naive cells by transfecting them with a GE Dharmacon GIPZ lentiviral vector containing the R8.74 helper plasmid, the VSVG envelope plasmid, and shRNA against ABCB1. The harvested lentiviruses were then transfected into adherent 293T naive cells. Three days after transfection, 293T transfected cells were evaluated for ABCB1 cell surface expression by flow cytometry (Biolegend, clone 4E3.16). As compared to 293T naive cells expressing low levels of ABCB1 endogenously, the transfected 293T cells did not express ABCB1. In addition, the GIPZ lentiviral vector contains GFP. All transduced 293T cells were GFP+, indicating that the transduction was successful along with a decrease in expression. The deficiency of ABCB1 expression was reconfirmed by flow cytometry in subsequent passages.

[0833] Generation of KO cell lines

[0834] To construct the ABCB1 and CD47 gene knockout HEK293 cell line, HEK293 host cells were first cultured by adherence in DMEM (Dulbeco Modified Eagle Medium, Gibco, Grand Island, NY, USA) supplemented with 10% (v / v) FBS and glutamine. Cells were cultured at 37°C with 5% CO2 at saturated humidity.

[0835] gRNA design was performed using the online CHOPCHOP web development tool to select target sites for CRISPR / Cas9, CRISPR / Cpf1, or TALEN-related mutagenesis. (Kornel Labun et al., (2016). Nucleic Acids Research; and Tessa G. Montague et al., (2014) Nucleic Acids Res . 42:W401-W407). All designed gRNAs were chemically synthesized (ThermoFisher).

[0836] Transfection of 293T cells was performed by lipid-based transfection using CRISPRMax reagent (ThermoFisher) according to the manufacturer's protocol. Briefly, one day prior to transfection, adherent cells were prepared at a rate of 0.2 x 10⁶ per well. 5 Canine cells were cultured in 96-well plates. On the day of transfection, solutions of GeneArt Platinum Cas9 protein, gRNA, and transfection reagents were added to the cells. At 72 h after transfection, cell culture was continued in the 96-well plate format for 2 weeks following the selection of single cells by the restriction dilution method. Subsequently, the selected clones were subcultured into 24-well plates and tested by genotyping using the Guide-it kit (Takara) according to the manufacturer's protocol. The genomic region surrounding the CRISPR target site for each gene was PCR amplified to determine whether gene editing resulted in insertion-deletion (indel) in one allele (monolithic) or two alleles (diolic) in the single isolated clone. The expression of the protein of interest on clones with mutations in both alleles was tested by FACS.

[0837] Composition of human-mouse sequences of tested molecules (human Fc, mouse Fvs)

[0838] Expression Vector: To generate antibody expression vectors, variable regions of the heavy and light chain DNA sequences were subcloned in-frame into the human IgG1 constant heavy chain or human IgG1 kappa constant light chain, which were pre-inserted into respective comprehensive 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) rapid early promoter for high-level gene expression. Two antibody chains were cloned into two different vectors.

[0839] The N-terminal signal sequences of mouse IgG heavy chains and kappa light chains were used for the secreted expression of the heavy and light chains, respectively. The signal peptides were cleaved during expression to leave the intact N-terminus. In the Fab construct, the C-terminus of the CH1 IgG1 constant region was fused with a 6K His tag to facilitate purification.

[0840] For the generation of bispecific antibody vectors, IgG1-derived bispecific molecules comprise at least two antigen-binding moieties capable of specifically binding to two distinct targets, Pgp (ABCB1) and CD47. The antigen-binding moieties are Fab fragments composed of heavy and light chains, each comprising variable and constant regions. A common light chain capable of pairing as both Fab anti-Pgp (aPgp) and Fab anti-CD47 (aCD47) and influencing acceptable binding was identified; its use enabled the prevention of LC pairing mispairing (see, e.g., U.S. Patent No. 8,765,412 (the full disclosure thereof is incorporated herein by reference)). Bispecific constructs were prepared based on electrostatic steering effects. (See, for example, Gunasekeran et al, (2010) Journal of Biological Chemistry 285, 19637-19646 (the full text of which is incorporated herein by reference). Briefly, polypeptide chains or half antibodies against a target are assembled as bispecific antibodies through charge pair substitution in the CH3 domain; one heavy chain contains K392D and K409D substitutions and the other contains E356K and D399K substitutions.

[0841] Figure 2 provides a diagram of a bispecific mAb containing sections containing the sequences A, 15D3; B, 5F9 on human IgG1Fc, along with C, a common light chain kappa sequence derived from MRK16.

[0842] The previously occurring monoclonal antibodies 15D3 (see, e.g., U.S. Patent 5,959,084 (the full contents of which are incorporated herein by reference)) and MRK16 (Iwahashi et al., Cancer Research 53, 1993 (the full contents of which are incorporated herein by reference)) were cloned into a human IgG1 / kappa expression vector as recombinant antibodies.

[0843] Variable heavy and light chain fragments of the mouse hybridoma sequence were made available and cloned into the same background as the leading sequence and invariant region.

[0844] Claim CD47 (5F9; see, e.g., U.S. Patent No. 9,017,675 (the full text of which is incorporated herein by reference)) antibody variable heavy chain and light chain fragments were cloned in two separate vectors on the same background of the leading sequence and invariant region.

[0845] Many combinations of these and other heavy and light chain fragments were generated and tested. Most of the tested constructs did not produce acceptable binding or activity. Examples of the antibodies constructed and tested, as well as their binding and cytotoxic properties, are provided in Fig. 8.

[0846] Figure 8 provides a comparison of binding and enhanced cell killing by different bispecific antibodies (comparison with various humanized or chimeric heavy or light chain combinations). Specifically, Column 1 ("Ab") of Figure 8 provides, for example, various bispecific formats tested in Column 1: 15D3 IgG1 DD / 5F9 IgG1 KK / MRK16 represents i) the 15D3 heavy chain sequence with the DD CH3 mutation described herein (chimeric in a human IgG1 framework), ii) the 5F9 heavy chain sequence with the KK CH3 mutation described herein, and iii) the MRK16 light chain (chimeric in a human IgG kappa framework). All light chains shown are in the human kappa format. Conjugation (Fig. 8, col. 2-3; "Kd (nM)" and "Bmax") and killing (Fig. 8, col. 3; "Killing") were determined as follows: "Kd (nM)", conjugation by ELISA (solid phase with Fc-tagged CD47 as the capture layer on the plate); "Bmax", conjugation of Bmax to the plate described for "Kd (nM)"; "Killing", paclitaxel titration (20 μM - 10 -8 Enhanced 293 Naive cell-killing (sensitization) in the presence of (μM range); "-" = 0, "±" = 0 to 0.5 log shift (in the 50% cell-killing curve), "+" = 1 to 2 log shift, "++" ≥ 2 log shift. 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 antibody binding to the indicated cell lines compared to the unconjugated control antibody: "-" = ~zero, "±" = 0 to ~0.5 log shift, "+" = ~1 log shift, "++" = 1 to >2 log shift (see Fig. 3A as an example of the unconjugated control and conjugated antibody).

[0847] It is important to note that the antibodies configured and tested above exhibit unacceptable binding characteristics or inefficient killing of target cells expressing both MDR1 and CD47, particularly compared to the main candidate.

[0848] For example, as shown in Figure 8, an antibody ("15D3 IgG1 DD / 5F9 IgG1 KK / MRK16") containing the 15D3 and 5F9 heavy chain charge-to-charge exchanged Fc region and a common MRK16-derived light chain was tested and exhibited desirable binding characteristics and produced enhanced killing (++) of target cells expressing both MDR1 and CD47. Therefore, this antibody was selected as the primary candidate. In comparison, when the common light chain of the primary candidate (i.e., "MRK16") was substituted with an alternative common light chain, such as "UIC2" (e.g., see "15D3 IgG1 DD / 5F9 IgG1 KK / UIC2") or "9F11" (e.g., see "15D3 IgG1 KK / 5F9 IgG1 DD / 9F11"), specific killing of cells expressing both CD47 and MDR1 was not observed. In addition, substituting an alternative anti-MDR1 heavy chain, such as "MM4.17 2" or "UIC2" heavy chain for the "15D3" heavy chain also resulted in significantly reduced specific killing of cells expressing both CD47 and MDR1 (see, e.g., Fig. 8, e.g., "MM4.17 2 IgG1 DD / 5F9 IgG1 KK / MRK16" and "UIC2 IgG1 KK / 5F9 IgG1 DD / MRK16").

[0849] The effect of exchanging the light chains used on antigen-expressing antibody-binding cells (i.e., "LC shuffle") was evaluated by ELISA and FACS binding assays. To test LC shuffle with CD47-specific heavy chains, a modified anti-CD47 heavy chain (5F9) was combined with M89, 15D3, MRK16, or UIC2 light chains, and binding was evaluated. Results showing the effect of LC shuffle on the bispecific antibody binding of various anti-MDR1 molecules to CD47-Fc-coated plates by ELISA are provided in Table 2:

[0850] 5F9 / 5F9 5F9 / M89 5F9 / 15D3 5F9 / MRK16 5F9 / UIC2 Bmax 2.782 4.22 2.957 2.939 3.076 Kd (nM) 0.001968 1510 0.2571 0.1695 6.297

[0851] It is important to note that in the table above, a 100-fold decrease in affinity was observed in the 5F9 / MRK16 combination compared to the unexchanged 5F9 control. Antibodies with Kd for CD47 similar to the 5F9 / MRK16 combination are desirable because they provide the essential degree of specificity when used in a bispecific format, so that the bispecific antibody exhibits low binding to normal cells and increased specificity against cancer cells expressing CD47 at higher levels than normal cells.

[0852] The results of the LC shuffle test showing the effect of combinations of the 15D3 heavy chain and the UIC2, MRK16, or 5F9 light chains on binding to CD47 as measured by ELISA are provided in Table 3:

[0853] 15D3 / 15D3 15D3 / UIC2 15D3 / MRK16 15D3 / 5F9 Bmax 3.202 3.449 3.935 3.095 Kd (nM) 10.9 168.6 229.1 251.9

[0854] In the table above, the remarkable affinity of 15D3 for CD47, which was reduced by ~20-fold after the combination of 15D3 HC and MRK16 LC compared to the unexchanged 15D3 control, is significant.

[0855] Candidate constructs with a combination of 15D3 and 5F9 heavy chains and a common MRK16 light chain exhibit acceptable binding and target antagonistic properties. In comparison, other tested heavy and light chain combinations showed unacceptable binding and / or target cell killing results when tested. Refer to Figure 8.

[0856] Purification of mAb

[0857] To purify the antibody format containing Fc, 10 μl of MabSelect™ SuRe™ (GE Healthcare) per 1 ml of supernatant was added to the harvested medium and stirred continuously overnight at 4°C. The next day, the protein A resin was applied to a 24-well filter plate using a vacuum manifold unit (Pall Lifesciences, USA). The resin was washed with PBS, the antibody was eluted in 50 mM phosphate at pH 3, and neutralized with 10x PBS at pH 13.

[0858] Histidine-tagged Fab was purified following the same procedure using Ni Sepharose 6 Fast Flow Histidine-Tagged Protein Purification Resin (GE Healthcare). The beads were washed with PBS, followed by washing with 25 mM phosphate buffer pH 7.4, 150 mM NaCl supplemented with 20 mM imidazole. The complex was eluted with 2 volumes of 25 mM phosphate buffer pH 7.4, 150 mM NaCl supplemented with 500 mM imidazole. Finally, the purified Fab was buffer-exchanged with PBS.

[0859] order

[0860] Pgp / MDR1 has the following amino acid sequence:

[0861]

[0862] The nucleic acid sequence encoding Pgp / MDR1 is available: P glycoprotein (ABCB1) (NM_000927) human genomic DNA Homo sapiens ATP binding cassette subfamily B member 1 (ABCB1), Ref Seq Gene on chromosome 7 (NG_011513 gen).

[0863] The nucleic acid sequence encoding CD47 is available: Homo sapiens CD47 molecule (CD47), transcript variant 1, mRNA NCBI Reference Sequence: NM_001777.3.

[0864] The variable heavy chain sequence of the anti-CD47 5F9 antibody is as follows:

[0865] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS.

[0866] The variable light chain sequence of the anti-CD47 5F9 antibody is as follows:

[0867] DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADVGVYYCFQGSHVPYTFGQGTKLEIK.

[0868] The variable heavy chain sequence of the anti-MDR1 MRK16 antibody is as follows:

[0869] EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA.

[0870] The sequence of the MRK16 antibody variable light chain is as follows:

[0871] DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK.

[0872] The antibody 15D3 sequence is available from U.S. Patent No. 5849877, and the antibody 15D3 heavy chain sequence is as follows:

[0873] EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSA,

[0874] The light chain sequence of antibody 15D3 is as follows:

[0875] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK.

[0876] The sequence of the anti-MDR1 UIC2 antibody light chain used as described herein is as follows:

[0877] DVVMTQTPRSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHIPPWTFGGGTKLDIK.

[0878] The sequence of the anti-MDR1 UIC2 antibody heavy chain used as described herein is as follows:

[0879] AVQLQQSGPELVKTGASVKISCKASGYSFSNYYIHWVKQSHGKSLEWIGFISCYNGATFYNQKFKGKATFTVDTSSSTAYMKFNSLTFEDSAVYYCARLPIQFGNFYPMDYWGQGTSVTVSS.

[0880] The sequence of the anti-MDR1 9F11 antibody light chain used as described herein is as follows:

[0881] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHRTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK.

[0882] The sequence of the anti-MDR1 9F11 antibody heavy chain used as described herein is as follows:

[0883] EVKLVESGGGLVKFGGSLKLSCAASGFTLSSYYMSWVRQSPEKRLELVAVINSNGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALYYCARPFYYSNSPFAYWGQGTLVTVSS.

[0884] The sequence of the heavy chain of the anti-MDR1 MM4.17 antibody used as described herein is as follows:

[0885] QVQLQESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVATISGGSYTYFPDSVKGRFTISRDNAKNTLYLQVSSLKSEDTAMYYCARPAEFRGYSWFAYWGQGTTVTVSS.

[0886] The sequence of the anti-MDR1 M89 antibody light chain used as described herein is as follows:

[0887] EIVLTQSPATLSLSPGERATLSCRASQSVGGSYLAWYQQKPGQAPRLLIYGASRRATGIPARFSGSGSGTDFTLTISSLQPEDFASYFCQQTNTFPLTFGGGTKVEIK.

[0888] The sequence of the anti-MDR1 M89 antibody heavy chain used as described herein is as follows:

[0889] QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQDPGKGLMWVSSISTDGSATKYADSVKGRFTISRDNAKNTVSLQMNSLRAEDTAVYYCVGGFLGWWGQGTLVTVSS.

[0890] result

[0891] Biophysical analysis tests on purified mAbs (GXII apparatus, reduced and non-reduced proteins)

[0892] The purity and monomer content of the final protein preparations were determined by high-throughput analysis on a Caliper LabChip GXII using the Protein Express LabChip Kit (Perkin-Elmer) as specified by the manufacturer. The chip was automatically primed on the instrument with a polymer solution containing 0.2% SDS and a fluorescent dye. The decolorized channels were filled with a polymer solution free of SDS and the dye. Briefly, proteins under reduced and non-reducing conditions were prepared by mixing small amounts (2–5 μL) of the sample with Caliper sample buffer containing or without DDT. The samples were denatured at 75°C for 5 minutes, centrifuged at 2000g for 3 minutes, and then run. Electrophoresis was generated using LabChip GXII Touch software (Perkin-Elmer).

[0893] Detection of CD47 and ABCB1 specific binding

[0894] The binding specificity of mAb, Fab, and bispecific IgG1-targeting CD47 was determined by ELISA using human CD47-Fc fusion protein (R&D systems). Briefly, microtiter plates were coated with 50 μl of purified human CD47-Fc fusion protein at 0.5 μg / ml in PBS, followed by blocking with 100 μl of 0.4% BSA in PBS. Dilutions of different antibody formats were added to each well in 1 / 3 sequential dilutions and incubated at room temperature for 1 hour. The known 5F9 anti-CD47 antibody was used as a positive control, and human IgG1 was used as an isotype control. The plates were subsequently washed three times with PBS / Tween and incubated at room temperature for 1 hour with HRP-conjugated donkey anti-human constant-specific secondary reagent. After washing, the plates were developed with HRP substrate. The reaction was stopped with 2M H2SO4, and the OD was measured at 520 nM.

[0895] The binding specificity of mAbs, Fabs, and bispecific IgG1 was tested by FACS using 293T cell lines naturally expressing CD47, 293T naive cells overexpressing the human ABCB1 target, 293T naive cells with ABCB1 knocked down using a lentiviral RNA vector, CD47 knockout 293T cells, and CD47 knockout 293T cells overexpressing human ABCB1. Briefly, different cell lines were incubated on ice for 1 hour with varying amounts of mAb, bispecific mAb, or human IgG1 isotype control antibodies. 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 samples were incubated on ice for an additional 1 hour. The sample was washed and analyzed using BD FACS Canto (BD Biosciences).

[0896] This example demonstrates the composition of a bispecific hetero-2 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 simultaneously present on the surface of a cell, the bispecific antibody binds to the cell with relatively high affinity / binding strength. In comparison, when MDR1 / Pgp or CD47 is absent or substantially reduced, the binding of the bispecific antibody is significantly reduced or undetectable (Fig. 4).

[0897] The bispecific antibody configured as exemplified in Fig. 2 contains one arm (arm A) that binds to and antagonizes a transporter protein (efflux pump Pgp), making the cell more sensitive to chemotherapy. At the same time, another arm (arm B), together with IgG1 Fc, binds to the "immuno-don't eat me" signal (CD47) on the cell surface, allowing for a more intense immune res...

Claims

Claim 1 A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and CD47, wherein the antibody molecule comprises two identical variable light chains (VL), a first variable heavy chain (VH), and a second VH chain, each VL chain comprising an antigen-binding site for MDR1, the first VH chain comprising an antigen-binding site for MDR1, and the second VH chain comprising an antigen-binding site for CD47, wherein the second VH chain binds to CD47 when paired with one of the VL chains, and the antigen-binding site of the first VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence No.: 9) (where X 2 is N), the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain having the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (Sequence No.: 17), the antigen-binding site of the second VL chain comprises light chain CDR 1-3 (LCDR 1-3) of the VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (Sequence No.:1) (where X 1 A bispecific antibody molecule that binds to cancer cells expressing both MDR1 and CD47 while exhibiting reduced binding to non-cancer cells expressing MDR1 and / or CD47. Claim 2 A bispecific antibody molecule according to claim 1, characterized in that two VL chains are humanized and / or comprise an amino acid sequence that is at least 90% identical to the following sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGNTYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIK (Sequence No. 8). Claim 3 A bispecific antibody molecule according to claim 1, characterized in that 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 (Sequence No. 16). Claim 4 A bispecific antibody molecule according to claim 1, characterized in that 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 (Sequence No.: 17), EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (Sequence 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, wherein the antibody molecule comprises two identical variable light chains (VL), a first variable heavy chain (VH), and a second VH chain, each VL chain comprising an antigen-binding site for MDR1, the first VH chain comprising an antigen-binding site for MDR1, the second VH chain comprising an antigen-binding site for PD-L1, the second VH chain binding to PD-L1 when paired with one of the VL chains, and the antigen-binding site of the first VH chain comprises a heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence No.: 9) (where X 2 is N), the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain having the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (Sequence No.: 32), a bispecific antibody molecule characterized in that the antigen-binding sites of the two VL chains comprise light chain CDR 1-3 (LCDR 1-3) of the VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (Sequence No.:1) (where X 1 is N). Claim 6 A bispecific antibody molecule that binds to multidrug resistance protein 1 (MDR1) and EGFR, wherein the antibody molecule comprises two identical variable light chains (VL), a first variable heavy chain (VH), and a second VH chain, each VL chain comprising an antigen-binding site for MDR1, the first VH chain comprising an antigen-binding site for MDR1, and the second VH chain comprising an antigen-binding site for EGFR, wherein the second VH chain binds to EGFR when paired with one of the VL chains, and the antigen-binding site of the first VH chain comprises the heavy chain CDR 1-3 (HCDR 1-3) of the VH chain having the following sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence No.: 9) (where X 2 is N), the antigen-binding site of the second VH chain comprises HCDR 1-3 of the VH chain having the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (Sequence No.: 36), a bispecific antibody molecule characterized in that the antigen-binding sites of the two VL chains comprise light chain CDR 1-3 (LCDR 1-3) of the VL chain having the following sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 1 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (Sequence No.:1) (where X 1 is N). Claim 7 A bispecific antibody molecule according to claim 1, characterized in that the bispecific antibody comprises an Fc domain modified to reduce or abolish the binding of the antibody to one or more Fcγ receptors. Claim 8 A bispecific antibody molecule according to claim 1, characterized in that the first VH chain is fused to the first Fc domain and the second VH chain is fused to the second Fc domain. Claim 9 A bispecific antibody molecule according to claim 8, characterized in that the Fc domain comprises a modified CH3 domain that preferentially forms a heterodimer comprising first and second VH chains. Claim 10 A bispecific antibody molecule according to claim 8, characterized in that the first and second Fc domains are human immunoglobulin G1 (IgG1) Fc domains. Claim 11 A pharmaceutical composition for treating cancer in a subject, comprising a bispecific antibody molecule of any one of claims 1 to 10 and a pharmaceutically acceptable excipient. Claim 12 A pharmaceutical composition according to claim 11, wherein the composition further comprises at least one additional active agent, and the at least one additional active agent comprises a chemotherapy agent, an inhibitor of a multidrug resistance transporter, an immunotherapy agent, or a combination thereof. Claim 13 A pharmaceutical composition according to claim 12, characterized in that at least one additional active agent is a chemotherapy agent. Claim 14 A pharmaceutical composition according to claim 13, characterized in that the chemotherapy agent is taxol, vinca alkaloid, or anthracycline. Claim 15 A pharmaceutical composition according to claim 11, characterized in that the subject has cancer determined to be resistant to treatment with a chemotherapy agent. Claim 16 A pharmaceutical composition according to claim 15, characterized in that the chemotherapy agent comprises paclitaxel, colchicine, verapamil, vinblastine, topotecan, doxorubicin, daunorubicin, etoposide, or nilotinib. Claim 17 One or more nucleic acids comprising one or more sequences encoding a bispecific antibody molecule of any one of claims 1 to 10. Claim 18 A mammalian cell isolated from one or more nucleic acids of claim 17, genetically modified. Claim 19 In claim 18, the isolated mammalian cell is characterized in that the cell is an immune cell. Claim 20 A kit for treating cancer in a subject, comprising: a bispecific antibody molecule of any one of claims 1 to 10 or a nucleic acid encoding said bispecific antibody molecule; and at least one additional activator. Claim 21 A pharmaceutical composition according to claim 11, characterized in that the cancer is drug-resistant or multidrug-resistant. 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