Anti-ABCG2 antibodies and their uses

Abstract

Antibodies targeting the cellular efflux pump ABCG2 are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of ABCG2 expression, the level of ABCG2 expression, and / or to inhibit ABCG2 function in cells, e.g., tumor cells, are also disclosed. Additionally, multispecific antibodies that bind to ABCG2 and polypeptides expressed on the surface of cancer cells are provided. The polypeptides can be the efflux pump MDR1 or cancer-associated antigens. Also provided are methods for treating a subject for cancer, comprising administering to the subject an anti-ABCG2 antibody disclosed herein, or a multispecific antibody targeting both ABCG2 and a cancer-associated antigen or MDR1.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 034,806, filed June 4, 2020, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listings provided as text files The Sequence Listing is provided herein as a text file "KNJY-004WO SEQ LIST_ST25.txt" having a size of 266 KB, created on May 24, 2021. The contents of the text file are incorporated herein by reference in their entirety. [Background technology]

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

[0004] Efflux pumps (EPs) are proteins expressed by living cells that have evolved to naturally efflux various compounds from cells. Members of the ATP-binding cassette (ABC) transporter family of proteins are examples of EPs that enable drug efflux. Although transporter structure varies from protein to protein (e.g., there are 49 known ABC family members in humans), they are all classified by the presence of two distinct domains: a highly conserved nucleotide-binding domain and a more variable transmembrane domain. Multidrug resistance protein 1 (MDR1), encoded by the ATP-binding cassette subfamily B member 1 (ABCB1) gene, was the first of these to be identified and has been extensively studied. ABCG2 expression increases in response to treatment with certain chemotherapeutic agents.

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

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

[0007] Antibodies that target the cellular efflux pump ABCG2 are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of ABCG2 expression, the level of ABCG2 expression, and / or to inhibit ABCG2 function in cells, e.g., tumor cells, are also disclosed. Additionally, multispecific antibodies that bind to cancer cells that overexpress ABCG2 and tumor-associated antigens (TAA) on the cell surface are provided. Additionally, multispecific antibodies that bind to cancer cells that overexpress ABCG2 and the efflux pump ABCB1 are provided. Also provided are methods for treating a subject for cancer, comprising administering to the subject an anti-ABCG2 antibody or multispecific antibody disclosed herein. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows a FACS analysis of the binding of anti-ABCG2 antibodies G2.65 and G.302 to HEK293 naive cells, HEK293 cells overexpressing human ABCG2 ("hG2"), and HEK293 cells overexpressing cynomolgus monkey ABCG2 ("cG2"). [Figure 2] 1 shows the characteristics of anti-ABCG2 antibodies. [Figure 3] 1 provides a schematic diagram of a bispecific antibody molecule comprising a first variable heavy chain A, a second variable heavy chain B, and a common light chain C. [Figure 4] Figure 1 shows binding of the indicated bispecific antibodies to 293T cells, 293T cells overexpressing KPB1 (KBP1 OX), and 293T cells overexpressing KPG2 (KBG2 OX). KPB1 refers to MDR1. KPG2 refers to ABCG2. The bispecific antibodies tested were: 15D3 IgG1 DD HC / G2.255KK HC / G2.255 LC, 15D3 IgG1 DD HC / G2.255KK HC / MRK16 LC, and 15D3 IgG1 DD HC / G2.255KK HC / 15D3 LC. Humanized versions of the bivalent, monospecific anti-ABCG2 antibodies were also tested. [Figure 5] FACS analysis showing that both ABCG2 and EGFR are expressed in A549 cells. [Figure 6] Binding analysis by FACS and ELISA of two different bispecific antibodies that bind to AGCG2 and EGFR. [Figure 7] Binding of the indicated anti-ABCG2 monoclonal antibodies to 293T cells overexpressing human ABCG2 ("hG2") and cynomolgus monkey ABCG2 (cG2), respectively, is shown along with the corresponding dissociation constants (Kd). [Figure 8] Binding of anti-ABCG2 antibody G2.748 to 293T cells overexpressing human or cynomolgus ABCG2 is shown in comparison to anti-ABCG2 antibody 5D3, along with the corresponding dissociation constants (Kd). [Figure 9] 9 to 11 show the binding of various recombinant anti-ABCG2 antibodies (Panels No. 1 to No. 3) to 293T cells overexpressing human and cynomolgus ABCG2, respectively, and the corresponding Kd values. [Figure 10] 9 to 11 show the binding of various recombinant anti-ABCG2 antibodies (Panels No. 1 to No. 3) to 293T cells overexpressing human and cynomolgus ABCG2, respectively, and the corresponding Kd values. [Figure 11] 9 to 11 show the binding of various recombinant anti-ABCG2 antibodies (Panels No. 1 to No. 3) to 293T cells overexpressing human and cynomolgus ABCG2, respectively, and the corresponding Kd values. [Figure 12] Figures 12 and 13 show the results of testing the listed recombinant anti-ABCG2 antibodies for efflux inhibitory activity according to the efflux blockade experimental procedure described herein, using the small molecule ABCG2 inhibitors fumitremordin C (FTC) and Ko143 as positive controls. [Figure 13] Figures 12 and 13 show the results of testing the listed recombinant anti-ABCG2 antibodies for efflux inhibitory activity according to the efflux blockade experimental procedure described herein, using the small molecule ABCG2 inhibitors fumitremordin C (FTC) and Ko143 as positive controls. [Figure 14] The effect of anti-ABCG2 antibodies G2.643, G2.420, and G.631 on topotecan cytotoxicity in 293T_ABCG2_OX cells, 293T cells stably transfected to express ABCG2, is shown using FTC and 5D3 as positive controls. [Figure 15] 1 shows the results of a xenograft study using topotecan-resistant Panc1 / T300 cells to test the efficacy of anti-ABCG2 antibodies G2.343, G2.636, and G2.629 alone and in combination with topotecan. Arrows indicate the dosing schedule. [Figure 16] 1 shows the results of a xenograft study testing the efficacy of anti-ABCG2 antibodies G2.343 and G2.631 alone and in combination with topotecan using the non-small cell lung cancer (NSCLC) epithelial cancer cell line A549 (ATCC, CCL-185). Arrows indicate the dosing schedule. [Figure 17] 1 shows the results of a xenograft study testing the efficacy of the anti-ABCG2 antibody G2.333 administered alone or in combination with camptothecin-11 (CPT11, irinotecan) using the non-small cell lung cancer (NSCLC) epithelial cancer cell line A549 (ATCC, CCL-185). Arrows indicate the dosing schedule. [Figure 18] Figure 1 shows the results of a xenograft study using topotecan-resistant Panc1 / T300 cells to test the efficacy of the bispecific anti-ABCG2 antibody G2.318 / KT3 / G2.318 administered alone or in combination with topotecan. Arrows indicate the dosing schedule. KT3 = cetuximab, an anti-EGFR antibody. [Figure 19] Figure 1 shows the results of a xenograft study using the HT1376 (ATCC, CRL-1472) bladder epithelial cancer cell line to test the efficacy of the bispecific anti-ABCG2 antibody G2.318 / KT9 / G2.318 administered alone or in combination with topotecan. Arrows indicate the dosing schedule. KT9 = atezolizumab, an anti-PL-L1 antibody. [Figure 20] 1 shows the efflux inhibitory activity and binding of various humanized G2.636 anti-ABCG2 antibodies to human and cynomolgus monkey ABCG2. [Figure 21] Schematic structures of two humanized ABCG2 / CD47 bispecific antibodies (5F9huscFv-G2.318.hu33 and B6H12huscFv-G2.318.hu33) and their binding to human and cynomolgus monkey ABCG2 are shown in comparison with G2.318.hu33 and 5D3, respectively. [Figure 22] Schematic structure of the humanized ABCG2 / HER2 bispecific antibody KT1scFv-G2.318.hu33 and its binding to human ABCG2 and human HER2. KT1=HER2. [Figure 23] Schematic structure of bispecific antibody G2.318KK KT9DD G2.318 and binding to ABCG2+KT9- (Figure 23A), ABCG2- KT9+ (Figure 23B), and ABCG2+ KT9+ 293T (Figure 23C) cells. KT9 = anti-PDL-1 monoclonal antibody atezolizumab. [Figure 24] The binding of the G2.643 antibody and its humanized form to human and cynomolgus ABCG2 (FIG. 24A) and the efflux inhibitory activity of the G2.643 antibody and its humanized form (FIG. 24B) are shown.

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

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

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

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

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

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

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

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

[0017] The term "binding" refers to a direct association between two molecules by ionic and / or hydrogen bonding interactions, including covalent interactions, electrostatic interactions, hydrophobic interactions, and interactions such as salt bridges and water bridges. ABCG2-specific antibodies specifically bind to an epitope within the ABCG2 polypeptide. The epitope can be a linear epitope formed by a contiguous stretch of amino acids, or a nonlinear or conformational epitope formed by a discontinuous stretch of amino acids. Nonspecific binding occurs at approximately 10 -7 Binding with an affinity of less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 This refers to binding based on affinity, such as M.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0047] Antibodies that bind to the cellular efflux pump ABCG2 are provided. Pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies are also provided. Methods of using the antibodies to detect the presence or absence of ABCG2 expression, the level of ABCG2 expression, and / or to inhibit ABCG2 function in cells, e.g., tumor cells, are also disclosed. Additionally, multispecific antibodies, such as bispecific antibodies, that bind to cancer cells that express both ABCG2 and a tumor-associated antigen (TAA) on their cell surface are provided. Additionally, bispecific antibodies that bind to cancer cells that express both ABCG2 and the efflux pump ABCB1 are provided. In certain embodiments, the bispecific antibodies disclosed herein bind to cells that express both antigens and do not detectably bind to cells that express only one of the antigens. Also provided are methods for treating a subject for cancer, comprising administering to the subject an anti-ABCG2 antibody or multispecific antibody disclosed herein.

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

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

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

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

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

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

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

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

[0056] antibody As summarized above, the present disclosure provides antibodies that bind to the cellular efflux pump ABCG2.

[0057] ABCG2, also known as CD388 and BCRP, is an energy-dependent efflux pump expressed from the ATP-binding cassette subfamily G member 2 (ABCG2) gene that is responsible for reduced drug accumulation in multidrug-resistant cells.

[0058] In some embodiments, the antibodies disclosed herein bind to one or more sites in the extracellular domain of ABCG2. In certain embodiments, the anti-ABCG2 antibodies of the present disclosure bind to human ABCG2. In certain embodiments, the anti-ABCG2 antibodies of the present disclosure bind to human ABCG2 expressed on the cell surface of human cells, e.g., cancer cells. In certain embodiments, the antibodies disclosed herein bind to one or more sites in the extracellular domain (ECD) of human ABCG2, and the ECD may comprise the sequence: KNDSTGIQNRAG (SEQ ID NO: 1), which corresponds to amino acid residues 417-428 of the human ABCG2 sequence with accession number NP_004818. In certain embodiments, the antibodies disclosed herein bind to one or more sites in the extracellular domain (ECD) of human ABCG2, and the ECD may comprise the sequence: LKPKADAF (SEQ ID NO: 2), which corresponds to amino acid residues 499-506 of the human ABCG2 sequence with accession number NP_004818. In certain embodiments, the antibodies disclosed herein bind to one or more sites in the extracellular domain (ECD) of human ABCG2, and the ECD may comprise the sequence: NLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATGNNPCNYATCTGEEYLVKQGIDLSPWGLWKNH (SEQ ID NO: 3), which corresponds to amino acid residues 557 to 630 of the human ABCG2 sequence having accession number NP_004818.

[0059] The antibodies of the present disclosure are i) the property of inhibiting efflux from ABCG2; ii) the property of increasing the sensitivity of cancer cells to treatment with a chemotherapeutic agent, thereby reducing the IC50 of the chemotherapeutic agent by at least two-fold; iii) the property of binding to human and cynomolgus monkey ABCG2; iv) being effective in an in vivo cell killing assay; v) being effective in inhibiting tumor growth even in the absence of chemotherapy; vi) preferential binding to ABCG2 mutants constrained in an open conformation; vii) having a lower range of affinity for ABCG2, such that it binds to cancer cells that express ABCG2 at higher levels compared to non-cancer cells, and binds to non-cancer cells at significantly lower levels; and viii) preferential binding to ABCG2 mutants constrained in a closed conformation; may have one or more of:

[0060] In certain embodiments, antibodies of the present disclosure may have a lower EC50 than the anti-ABCG2 antibody 5D3. As used herein, EC50 refers to the concentration of antibody that provides half of the maximal response (e.g., half of the maximal fluorescence intensity). Antibodies of the present disclosure may have an EC50 of 100 nM or less, e.g., 100 nM to 4 nM, 80 nM to 4 nM, 60 nM to 4 nM, 40 nM to 4 nM, 30 nM to 4 nM, 20 nM to 4 nM, 15 nM to 4 nM, or 10 nM to 4 nM. The EC50 of a test antibody may be determined by flow cytometry or ELISA. For example, flow cytometry may involve contacting cells expressing ABCG2 (e.g., human wild-type ABCG2 or mutant ABCG2) with an antibody (the antibody is serially diluted) in flow cytometry buffer and incubating at room temperature or 4°C for a time sufficient for the antibody to bind to the cells (e.g., 10 minutes to 1 hour). After incubation, the cells may optionally be washed to remove nonspecifically bound antibody and / or contacted with a fluorescently labeled secondary antibody that specifically binds to the test antibody. After incubation, the fluorescently labeled secondary antibody may be removed and the cells may be washed. The washed cells may be sorted by flow cytometry, and the number of cells bound to the fluorescently labeled secondary antibody may be counted. The concentration providing a half-maximal response (e.g., half-maximal fluorescence intensity) is measured as the EC50. In a variation of the flow cytometry assay, the cells may be 293T cells overexpressing ABCG2. In certain embodiments, the antibodies of the present disclosure may have an EC50 higher than the EC50 of the anti-ABCG2 antibody 5D3.

[0061] In certain embodiments, in addition to having one or more of the properties i)-viii) listed above, one or more of the anti-ABCG2 antibodies of the present disclosure may have an EC50 that is at least half of the EC50 of the anti-ABCG2 antibody 5D3. In certain embodiments, in addition to having one or more of the properties i)-viii) listed above, one or more of the anti-ABCG2 antibodies of the present disclosure may have an EC50 that is at least twice the EC50 of the anti-ABCG2 antibody 5D3.

[0062] The IC50 of a test antibody can be determined by measuring inhibition of cell proliferation. The IC50 can be measured by using the test antibody alone to determine the concentration of antibody that produces a half-maximal response. The IC50 of a chemotherapeutic agent can be measured in the absence and presence of the test antibody to determine the effect of the antibody on the IC50 chemotherapeutic agent. The chemotherapeutic agent can be topotecan. The cells can be a cancer cell line. The cancer cell line can be N6 / ADR, a doxorubicin-selected B1-positive variant of NALM6, a human acute lymphoblastic leukemia (ALL) cell line. N6 / ADR cells are also referred to as NALM6 / ADR cells. When determining the IC50 of an antibody, the cells can be contacted with the antibody alone, and the antibody is tested in serial dilutions. The cells can be contacted with the antibody and a chemotherapeutic agent to determine the effect of the antibody on the IC50 of the agent, and the agent is tested in serial dilutions. The cells may be incubated at 37°C for a period of time (e.g., 24 to 84 hours), and cell viability may be assessed using standard reagents and methods. The antibodies disclosed herein may sensitize cancer cells to treatment with a chemotherapeutic agent, thereby reducing the IC50 of the chemotherapeutic agent by at least 5-fold. The cancer cells may be N6 / ADR. The chemotherapeutic agent may be topotecan. In certain embodiments, the antibodies of the present disclosure may reduce the IC50 of the chemotherapeutic agent by 5-fold or more, e.g., 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, e.g., 5-fold to 10-fold.

[0063] In certain embodiments, one or more of the anti-ABCG2 antibodies disclosed herein bind to both human and cynomolgus monkey ABCG2, a property that can be utilized to determine the safety of the antibody in animal models.

[0064] In certain embodiments, the anti-ABCG2 antibodies disclosed herein are specific for ABCG2 and do not exhibit significant binding to other antigens.

[0065] In certain embodiments, the in vitro cell killing activity of the antibodies of the present disclosure may be greater than that observed with the 5D3 antibody. For example, the antibodies of the present disclosure may have in vitro cell killing activity that is two-fold or more greater than that of the 5D3 antibody.

[0066] In certain embodiments, one or more of the antibodies provided herein preferentially bind to an ABCG2 variant constrained in an open conformation. The ABCG2 variant can be human or cynomolgus ABCG2 containing the substitution E211Q, where the numbering of amino acid positions refers to human ABCG2. Such an antibody can bind to an ABCG2 variant constrained in an open conformation with at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more) greater affinity than wild-type ABCG2 or an ABCG2 variant constrained in a closed conformation.

[0067] In certain embodiments, one or more of the antibodies provided herein preferentially bind to an ABCG2 mutant constrained in a closed conformation. The ABCG2 mutant can be human or cynomolgus ABCG2 containing the following substitutions: (i) K86M and S87A, (ii) K86M, S87A, and Q126A, or (iii) K86M, S87A, Q126A, R246E (amino acid position numbering refers to human ABCG2). Such an antibody can bind to an ABCG2 mutant constrained in a closed conformation with at least 2-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more) greater affinity than wild-type ABCG2 or an ABCG2 mutant constrained in an open conformation.

[0068] In certain embodiments, the antibodies provided herein are monospecific bivalent anti-ABCG2 antibodies. In certain embodiments, the monospecific bivalent anti-ABCG2 antibodies of the present disclosure do not contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 HCDRs 1-3 and LCDRs 1-3 present in the anti-ABCG2 antibody 5D3.

[0069] The 5D3 antibody has a variable heavy chain with the sequence: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 4); and a variable light chain having the sequence: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIK (SEQ ID NO: 5).

[0070] HCDR1 to 3 and LCDR1 to 3 of the 5D3 antibody, as defined according to the Kabat nomenclature, are as follows: HCDR 1: GFSITSDYAW (SEQ ID NO: 6); HCDR 2: GYINFDGGTTYNPSLRG (SEQ ID NO: 7); HCDR 3: ATFYGAKGTLDY (SEQ ID NO: 8); LCDR 1: KASGYILNRLA (SEQ ID NO: 9); LCDR 2: GATSLET (SEQ ID NO: 10); LCDR 3: QQYWSTPWT (SEQ ID NO: 11).

[0071] In some embodiments, one or more of the subject antibodies, when bound to cells expressing ABCG2, may interfere with the function of cellular ABCG2 protein. Thus, one or more antibodies of the present disclosure may inhibit efflux by ABCG2 protein, e.g., 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 ABCG2 in the absence of the subject antibody. In some embodiments, the subject antibodies, when bound to cells expressing ABCG2, may interfere with the action of ABCG2 by other mechanisms, e.g., by making ABCG2 leaky, which in turn enhances the uptake of chemotherapeutic agents and / or reduces cell viability.

[0072] In certain embodiments, an antibody that binds to ABCG2 is provided, which competes for binding to ABCG2 with an antibody comprising the heavy chain complementarity determining region (HCDR) and light chain CDR (LCDR) of the pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. For example, in one embodiment, an anti-ABCG2 antibody of the present disclosure competes for binding to ABCG2 with the G2.302 antibody listed in Table 2. In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are defined according to the Kabat nomenclature.

[0073] In certain embodiments, the antibody comprises HCDR1, HCDR2, and HCDR3 of the VH region of an antibody listed in Table 2. In certain embodiments, HCDR1, HCDR2, and HCDR3 are defined according to Kabat nomenclature. For example, in one embodiment, an anti-ABCG2 antibody of the disclosure that competes with the G2.302 antibody listed in Table 2 for binding to ABCG2 comprises HCDR1, HCDR2, and HCDR3 of the VH region of the G2.302 antibody.

[0074] Any suitable approach can be used to determine whether a first antibody competes with a second antibody for binding to ABCG2. Whether a first antibody "competes" with a second antibody for binding to a compound can be easily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, through antibody competition assays. For example, a sample of a first antibody can be bound to a solid support. A sample of a second antibody that is thought to be able to compete with the first antibody is then added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies bind to separate, distinct sites on the compound, the labeled antibody will bind at the same level regardless of the presence or absence of a competing antibody. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete, reducing the amount of labeled antibody that binds to the antigen. If an excess of unlabeled antibody is present, very little, if any, labeled antibody will bind.

[0075] For purposes of this disclosure, a competing antibody is one that reduces antibody binding to a compound by about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more. Details of procedures for performing such competitive assays are well known in the art and can be found, for example, in Harlow and Lane, *Antibodies*, *A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, pp. 567-569, 1988, ISBN 0-87969-314-2. Such assays can be quantified using purified antibodies. A standard curve can be established by titrating one antibody against itself; i.e., the same antibody is used as both the label and the competitor. The ability of an unlabeled competing antibody to inhibit the binding of the labeled antibody to its target epitope is titrated. The results can be plotted and the concentrations required to achieve the desired degree of binding inhibition can be compared.

[0076] In certain embodiments, an antibody that specifically binds to ABCG2 comprises (i) HCDRs 1-3 and light chain CDRs (LCDRs 1-3) of a pair of variable heavy (VH) and variable light (VL) chain regions of an antibody listed in Table 2, (ii) HCDRs 1-3 of a VH region of an antibody listed in Table 2, or (iii) LCDRs 1-3 of a VH region of an antibody listed in Table 2. HCDRs and LCDRs may be defined based on the Kabat nomenclature.

[0077] In certain embodiments, antibodies of the present disclosure that specifically bind to human ABCG2 comprise the HCDR1, HCDR2, and HCDR3 sequences, and the LCDR1, LCDR2, and LCDR3 sequences of the antibodies listed in Table 2. In addition to binding to human ABCG2, one or more of the antibodies provided herein may bind to ABCG2 from other mammalian species, such as mouse, monkey, chimpanzee, etc. The antibodies may be made in mouse or rat. Table 2 lists the animals in which the antibodies were raised. Some of the antibodies have been humanized. Table 2: From left to right, column 1: anti-ABCG2 antibody name, column 2: VH region, column 3: HCDR1, column 4: HCDR2, column 5: HCDR3, column 6: VL region, column 7: LCDR1, column 8: LCDR2, column 9: LCDR3. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] Table 2-7 Table 2-8 Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 Table 2-32 Table 2-33

[0078] The anti-ABCG2 antibodies listed in Table 2 are also referred to as anti-KPG2 antibodies and can be referred to by the antibody numbers listed in Table 2.

[0079] The term "antibody molecule" encompasses antibodies, as defined herein, including antigen-binding fragments thereof. In certain embodiments, an antibody molecule comprises two variable light chains (VL) and two variable heavy chains (VH). In certain embodiments, an antibody molecule further comprises heavy and light chain constant regions. The heavy and light chain constant regions may be from a human antibody, such as a human IgG1 antibody. The human IgG1 heavy chain (HC) constant region may be modified to contain mutations that reduce antibody-dependent cellular cytotoxicity (ADCC). Additionally, or alternatively, the two VH chains may each be conjugated to a different human IgG1 HC constant region, with each human IgG1 HC constant region having substitutions that favor dimer formation between the different human IgG1 HC constant regions. Such HC regions are described in further detail herein. In certain embodiments, when the antibody molecule is a bispecific antibody molecule, one of the human IgG1 HC constant regions may contain substitutions introducing one or more amino acids with positively charged side chains, and the other human IgG1 HC constant region may contain substitutions introducing one or more amino acids with negatively charged side chains that favor the formation of dimers between the two different HCs.

[0080] In certain embodiments, the antibody molecule comprises HCDR1-3 and / or LCDR1-3 of a pair of VH and VL regions of an antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDR1-3 and / or LCDR1-3 of the G2.248 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDR1-3 and / or LCDR1-3 of the G2.255 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDR1-3 and / or LCDR1-3 of the G2.256 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDR1-3 and / or LCDR1-3 of the G2.65 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDR1-3 and / or LCDR1-3 of the G2.30 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.173 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.333 antibody, G2.343 antibody, G2.636 antibody, G2.629 antibody, G2.643 antibody, G2.420 antibody, or G2.631 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.636 antibody listed in Table 2. In one embodiment, the antibody molecule comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.318 antibody listed in Table 2.

[0081] In certain embodiments, the antibody comprises HCDR1-3 and / or LCDR1-3 of a pair of VH and VL regions of an antibody listed in Table 2, and reduces the IC50 of a chemotherapeutic agent by 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, e.g., 5-fold to 10-fold.

[0082] The EC50 can be the antibody concentration that produces a 50% maximal response (e.g., the response is the binding of the antibody to its antigen). In one embodiment, the antibody has an EC50 of 100 nM or less and / or an EC50 that is at least half of the EC50 of an anti-ABCG2 antibody, such as 5D3, and comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.248 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less and / or an EC50 that is at least half of the EC50 of an anti-ABCG2 antibody, such as 5D3, and comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.255 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less and / or an EC50 that is at least half of the EC50 of an anti-ABCG2 antibody, such as 5D3, and comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.256 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less and / or an EC50 that is at least half of the EC50 of an anti-ABCG2 antibody such as 5D3, and comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.65 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less and / or an EC50 that is at least half of the EC50 of an anti-ABCG2 antibody such as 5D3, and comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.30 antibody listed in Table 2. In one embodiment, the antibody has an EC50 of 100 nM or less and / or an EC50 that is at least half of the EC50 of an anti-ABCG2 antibody such as 5D3, and comprises HCDRs 1-3 and / or LCDRs 1-3 of the G2.173 antibody listed in Table 2.

[0083] In some embodiments, the antibody comprises a VL region and a VH region that are present on separate polypeptides, while in other embodiments, the VL region and the VH region are comprised within a single polypeptide.

[0084] The antibodies of the disclosure may comprise a humanized light chain, a humanized heavy chain, or both. In certain embodiments, the antibody may be a humanized antibody comprising a VH region as shown in Table 2 for the G2.173humanized1, G2.173humanized1, or G2.173humanized1 antibody. In certain embodiments, the antibody may be a humanized antibody comprising a VL region as shown in Table 2 for the G2.173humanized1, G2.173humanized1, or G2.173humanized1 antibody. In certain embodiments, the antibody may be a humanized antibody comprising VH and VL regions as shown in Table 2 for the G2.173humanized1, G2.173humanized1, or G2.173humanized1 antibody.

[0085] The antibody of the present disclosure may be selected from the group consisting of an Ig monomer, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an scFv, a scAb, a dAb, and an Fv.

[0086] multispecific antibodies In certain embodiments, the antibodies of the present disclosure are multispecific, capable of binding epitopes present on two different target proteins. The number of different target proteins, and therefore the number of different epitopes, bound by a multispecific antibody can vary and can be two (i.e., bispecific), three (trispecific), four, or more.

[0087] In certain embodiments, an antibody of the present disclosure is a multispecific (e.g., bispecific) antibody capable of binding to at least two different epitopes, one of which is in ABCG2 (e.g., human ABCG2) and the other of which is in a tumor-associated antigen (TAA) expressed on the cell surface of cancer cells. In another embodiment, a multispecific antibody of the present disclosure binds to human ABCG2 and the efflux pump MDR-1. In certain embodiments, the VH and VL chains of a bispecific antibody of the present disclosure are selected such that the antibody binds to cells expressing both antigens, e.g., cancer cells, but exhibits substantially reduced binding to cells expressing only one of the antigens.

[0088] In certain embodiments, the VH and VL chains of the bispecific antibody of the present disclosure are selected so that the antibody binds to cells that overexpress both antigens and exhibits substantially lower binding to cells that express both antigens at normal levels or express or overexpress only one of the antigens. Such antibodies specifically bind to cancer cells that overexpress both antigens and therefore have minimal off-target effects due to reduced binding to normal cells. As used herein, the term overexpressed is meant to encompass expression levels higher than those detected in normal cells. For example, cancer cells that overexpress a TAA express a higher level of the TAA than the level of the TAA in normal cells of the same type as the cancer cells, such as epithelial cells. Normal cells may not express the TAA or may express a certain level of the TAA. Cancer cells may overexpress the TAA compared to the expression level in normal cells of the same type.

[0089] Tumor-associated antigens (also referred to herein as cancer-associated antigens) refer to antigens that are overexpressed in cancer cells compared to their expression levels in non-cancerous cells of the same type. For example, a TAA is an antigen that is not expressed at detectable levels in normal cells but is expressed in cancer cells, where the normal cells and cancer cells are the same cell type, e.g., epithelial cells. In other embodiments, a TAA is an antigen that is expressed in normal cells but at higher levels in cancer cells. A TAA can be expressed on the cell surface of mammalian cancer cells. In certain embodiments, the TAA can be CD47, PDL1, erbB-1, erbB-2, or EGFR. A tumor-associated antigen can be a neoantigen. Neoantigens are a class of tumor antigens that arise from tumor-specific mutations that change the amino acid sequence of the encoded protein compared to the amino acid sequence of the unmutated protein.

[0090] In certain embodiments, the bispecific antibody specifically binds to cancer cells that overexpress both ABCG2 and the efflux pump MDR-1 or a cancer-associated antigen, hi certain embodiments, the bispecific antibody binds to cancer cells that overexpress both ABCG2 and MDR-1 and shows substantially lower binding to cells that do not overexpress both ABCG2 and MDR-1.

[0091] In certain embodiments, the bispecific antibody specifically binds to cancer cells that overexpress both ABCG2 and a TAA. In certain embodiments, the bispecific antibody binds to cancer cells that overexpress both ABCG2 and a TAA and shows substantially lower binding to cells that do not overexpress both ABCG2 and a TAA.

[0092] In certain embodiments, bispecific antibodies of the present disclosure can be selected based on binding to cells that express both ABCG2 and a TAA or MDR-1 at levels two-fold or more (e.g., at least three-fold, four-fold, five-fold, ten-fold or more) than expressed by normal cells, and exhibit substantially lower binding to normal cells or cells that overexpress only one of ABCG2 and a TAA or MDR-1.

[0093] In certain embodiments, the bispecific antibody increases the sensitivity of cancer cells to treatment with a chemotherapeutic agent, thereby reducing the IC50 of the chemotherapeutic agent when administered with the multispecific antibody by at least two-fold compared to the IC50 of the chemotherapeutic agent when administered with the anti-ABCG2 monospecific bivalent antibody. The IC50 can be measured by methods as provided herein. The chemotherapeutic agent can be topotecan. The cancer cells can be drug-resistant cancer cells. In certain embodiments, the multispecific antibody of the present disclosure can reduce the IC50 of the chemotherapeutic agent by five-fold or more, e.g., six-fold or more, seven-fold or more, eight-fold or more, nine-fold or more, or ten-fold or more, e.g., five to ten-fold.

[0094] In certain embodiments, the bispecific antibody may have in vivo cell killing activity, e.g., reduction of tumor volume, even in the absence of administration of a chemotherapeutic agent such as topotecan.

[0095] In some embodiments, multispecific antibodies, e.g., bispecific antibodies, of the present disclosure can include a common light chain. As used herein, the term "common light chain" generally refers to the use and incorporation of two copies of the same light chain into a multispecific antibody. In other words, the light chain in the assembled multispecific antibody associates with an ABCG2-specific heavy chain, and a second copy of the same light chain associates with a TAA-specific heavy chain or an MDR-1 antigen-specific heavy chain. The common light chain can be, for example, from an anti-ABCG2 antibody having a VL chain comprising LCDRs 1-3 of an antibody listed in Table 2, such as the G2.248, G2.255, G2.256, G2.65, G2.302, G2.173, G2.173.humanized 1, G2.173.humanized 2, or G2.173.humanized 3 antibodies. In other embodiments, the common light chain can be from an anti-MDR-1 antibody, such as MRK16 or 15D3. In other embodiments, the common light chain can be from an unrelated antibody, an antibody library, or a source of synthetically designed or in vitro generated antibodies. In another example, a bispecific antibody may not contain a common light chain, but instead contain a first heavy chain and a first light chain that binds to G2, and a second heavy chain and a second light chain that binds to another antigen.

[0096] Bispecific antibodies against ABCG2 and MDR1 In a specific embodiment, a bispecific antibody molecule that binds to ABCG2 and MDR1 may comprise two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain, wherein each VL chain comprises an antigen-binding site for MDR1, the first VH chain comprises an antigen-binding site for MDR1, and the second VH chain comprises an antigen-binding site for ABCG2, and the second VH chain binds to ABCG2 when paired with one of the light chains. In a specific embodiment, the second VH chain comprises HCDRs 1-3 of the VH chain of an anti-G2 antibody listed in Table 2, the second VH chain comprises HCDRs 1-3 of the VH chain of an anti-MDR1 antibody, such as 15D3, and the common light chain comprises LCDRs 1-3 of the VL region of another anti-MDR1 antibody, such as the anti-MDR1 antibody 15D3 or MRK16.

[0097] In a specific embodiment, the first VH chain of the bispecific antibody comprises HCDRs 1-3 of the VH chain from an anti-MDR1 antibody such as 15D3, where HCDR1 comprises the sequence: GFTFSRYTMS (SEQ ID NO: 419), HCDR2 comprises the sequence: VATISSGGGNTYYPDSVKG (SEQ ID NO: 362), VATISSGGGQTYYPDSVKG (SEQ ID NO: 363), or VATISSGGGSTYYPDSVKG (SEQ ID NO: 364), and HCDR3 comprises the sequence: ARYGAGDAWFAY (SEQ ID NO: 365). In a specific embodiment, the second VH chain of the bispecific antibody comprises HCDRs 1-3 of the VH chain of an anti-ABCG2 antibody having the sequence shown in Table 2. In a specific embodiment, the common VL chain of the bispecific antibody comprises LCDRs 1-3 of the VL chain of the anti-MDR1 antibody 15D3.

[0098] In certain embodiments, the second VH chain of the bispecific antibody comprises HCDRs 1-3 of the VH chain of G2.255, an anti-ABCG2 antibody listed in Table 2. In certain embodiments, the second VH chain comprises the amino acid sequence: EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQTPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVS (SEQ ID NO: 366).

[0099] In a particular embodiment, the two identical VL chains comprise LCDR1-3 of the VL chain of the anti-MDR1 antibody 15D3, having the amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367).

[0100] In certain embodiments, the two identical VL chains comprise LCDRs 1-3 of an anti-MDR1 antibody, (i) LCDR1 comprises the sequence: RSSQSIVHSTGNTYLE (SEQ ID NO: 368); (ii) LCDR2 comprises the sequence: KVSNRFS (SEQ ID NO: 305); (iii) LCDR3 comprises the sequence: QGSHFPRT (SEQ ID NO: 369).

[0101] In a particular embodiment, the two identical VL chains have the amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 2 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) (wherein X 2 is N, Q, or S).

[0102] In certain embodiments, the two identical VL chains comprise LCDRs 1-3 of an anti-MDR1 antibody, (i) LCDR1 has the sequence: RSSQSIVHSTGX 2 TYLE (SEQ ID NO: 371), (ii) LCDR2 comprises the sequence: KISNRFS (SEQ ID NO: 372); (iii) LCDR3 comprises the sequence: FQASHFPRT (SEQ ID NO: 373); where X 2 is N, Q, or S.

[0103] In certain embodiments, a subject antibody may comprise a VH chain comprising the HCDRs of a VH chain listed in Table 2 (e.g., the G2.255 antibody), and a VL chain comprising the LCDR of the MRK16 antibody or 15D3 antibody, or a humanized version thereof. The HCDRs and LCDRs may be defined according to the Kabat nomenclature.

[0104] The amino acid sequence of the VL chain of the MRK16 antibody is as follows: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 374)

[0105] The amino acid sequence of the humanized VL chain of the MRK16 antibody is as follows: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGX 2 TYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIKR (SEQ ID NO: 375) (where X 2 is N, Q, or S)

[0106] The VL chain LCDRs 1 to 3 have the following sequences: CDR1 (RSSQSIVHSTGX 2 TYLEW, SEQ ID NO: 376) (where X 2is N, Q, or S), CDR2 (KISNRFSG, SEQ ID NO: 377), and CDR3 (FQASHFPRTF, SEQ ID NO: 378).

[0107] In a particular embodiment, the VL chain has the MRK16 VL chain sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 374), or the sequence: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGX 2 TYLEWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIKR (SEQ ID NO: 375) (where X 2 is N, Q, or S).

[0108] In certain embodiments, the LCDR is selected from the group consisting of the 15D3 VL chain: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367).

[0109] The 15D3 LCDR1-3 sequences defined according to the Kabat nomenclature are as follows: 15D3 LCDR1: RSSQSIVHSTGNTYLE (SEQ ID NO: 368) 15D3 LCDR2: KVSNRFS (sequence number 305) 15D3 LCDR3: QGSHFPRT (SEQ ID NO: 369)

[0110] In certain embodiments, the VL chain may have a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including 100%) identical to the 15D3 VL chain sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367).

[0111] In certain embodiments, a subject antibody may comprise a VL chain comprising the LCDR of a VL chain listed in Table 2, and a VH chain comprising the HCDR of the MRK16 antibody or 15D3 antibody, or a humanized version thereof. The HCDRs and LCDRs may be defined according to the Kabat nomenclature.

[0112] The amino acid sequence of the VH chain of the MRK16 antibody is as follows: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA (SEQ ID NO: 379)

[0113] The amino acid sequence of the VH chain of the 15D3 antibody is as follows: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 380)

[0114] The amino acid sequence of the humanized VH chain of the 15D3 antibody is as follows: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGX 2TYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSA (SEQ ID NO: 381) (where X 2 is N, Q, or S)

[0115] The 15D3 HCDR1-3 sequences defined according to the Kabat nomenclature are as follows: 15D3 HCDR1: RYTMS (SEQ ID NO: 382), 15D3 HCDR2: TISSGGGX 2 TYYPDSVKG (SEQ ID NO: 383) (where X 2 is N, Q, or S), 15D3 HCDR3: YGAGDAWFAY (SEQ ID NO: 384)

[0116] In certain embodiments, the VH chain has the 15D3 VH chain sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 380), or the sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSSA (SEQ ID NO: 381) (where X 2 is N, Q, or S).

[0117] In certain embodiments, the second VH chain comprises a humanized version of the VH chain of an antibody listed in Table 2, wherein the second VH chain comprises a humanized version of the VH chain of the anti-MDR1 antibody 15D3, and the humanized 15D3 VH chain has the sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 385), EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 386), or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQAPGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSLYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (sequence number 387), and the common light chain comprises LCDR1 to LCDR3 of the VL region of the anti-MDR1 antibody 15D3.

[0118] In certain embodiments, the bispecific antibody binds to MDR1 and ABCG2, reduces the IC50 of a chemotherapeutic agent by 2-fold or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10-fold, or more), and comprises a common light chain, a VH chain comprising an HCDR from a VH chain listed in Table 2, and a VH chain comprising an HCDR from an anti-MDR1 antibody such as 15D3. The common light chain can comprise an LCDR from a VL chain listed in Table 2 (e.g., the VL of the antibody in Table 2 from which the VH HCDR is derived), or from an anti-MDR1 antibody such as MRK16 or 15D3. In certain embodiments, the chemotherapeutic agent is topotecan.

[0119] Bispecific antibodies against ABCG2 and TAA Also provided herein is a bispecific antibody having at least one or more of the above-described properties. In a specific embodiment, the bispecific antibody molecule of the present disclosure binds to ATP-binding cassette subfamily G member 2 (ABCG2) and a tumor-associated antigen (TAA), wherein the antibody molecule comprises two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain, wherein the VL chains each comprise an antigen-binding site for ABCG2, the first VH chain comprises an antigen-binding site for ABCG2, and the second VH chain comprises an antigen-binding site for the TAA, and the second VH chain binds to the TAA when paired with one of the light chains; or wherein the VL chains each comprise an antigen-binding site for the TAA, the first VH chain comprises an antigen-binding site for ABCG2, and the second VH chain comprises an antigen-binding site for the TAA, and the first VH chain binds to ABCG2 when paired with one of the light chains. The TAA can be CD47, PDL1, EGFR, erbB-1, or erbB-2.

[0120] In a particular embodiment, the VL chains each comprise an antigen-binding site for ABCG2, the first VH chain comprises an antigen-binding site for ABCG2, and the second VH chain comprises an antigen-binding site for a TAA, and the second VH chain binds to the TAA when paired with one of the light chains.

[0121] In certain embodiments, the first VH chain comprises heavy chain complementarity determining regions 1-3 (HCDRs 1-3) of an anti-ABCG2 antibody listed in Table 2.

[0122] In a specific embodiment, the first VH chain comprises heavy chain complementarity determining regions 1-3 (HCDR1-3), wherein HCDR1 comprises the sequence: DDYVH (SEQ ID NO: 85), HCDR2 comprises the sequence: RIDPANGNTRYAPKFRG (SEQ ID NO: 115), and HCDR3 comprises the sequence: PLWVGGFAY (SEQ ID NO: 157), or the first VH chain comprises the amino acid sequence: QVQLQQSGADLVRPGASVKLSCTASGFNIKDDYVHWVKQRPEQGLEWIGRIDPANGNTRYAPKFRGKATMTADTSSNTAYLQLSSLTSADTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 16), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPANGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 17), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPAQGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 18), or It comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPASGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 19).

[0123] In some embodiments, the antigen-binding sites of the two VL chains comprise the light chain CDRs 1-3 (LCDRs 1-3) of an antibody listed in Table 2.

[0124] In some embodiments, the antigen binding site of the two VL chains comprises an LCDR1 comprising the sequence: RSSQSLVHSDVNTYLH (SEQ ID NO: 270), an LCDR2 comprising the sequence: KVSNRFS (SEQ ID NO: 305), and an LCDR3 comprising the sequence: SQTTHVPYT (SEQ ID NO: 334), or the VL chain comprises the amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDVNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVESEDLGIYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 199), or DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDVNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 200).

[0125] In a particular embodiment, the first VH chain comprises heavy chain complementarity determining regions 1-3 (HCDR1-3), wherein HCDR1 comprises the sequence: SGYIS, HCDR2 comprises the sequence: WIYAGTGISNFNQKFTG, and HCDR3 comprises the sequence: GARKTLDF, or the first VH chain comprises the amino acid sequence: It comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to QGQMHQSGAELVKPGASVKLSCKTSGFTFNSGYISWLKQKPRQSLEWIAWIYAGTGISNFNQKFTGKAQLTVDTSSSTAYMQLSSLTSADSAIYFCASGARKTLDFWGQGTSVTVSS (SEQ ID NO: 15).

[0126] In certain embodiments, the antigen-binding sites of the two VL chains comprise the light chain CDRs 1-3 (LCDRs 1-3) of the antibodies listed in Table 2.

[0127] In a particular embodiment, the antigen binding sites of the two VL chains comprise an LCDR1 comprising the sequence: G2.65 KASDQINYWLA (SEQ ID NO: 269), an LCDR2 comprising the sequence: GATSLET (SEQ ID NO: 10), and an LCDR3 comprising the sequence: QQYWTTPYT (SEQ ID NO: 333), or the VL chain comprises the amino acid sequence: DIQMTQSSSYLSVSVGGRVTITCKASDQINYWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSFQTEDVATYYCQQYWTTPYTFGGGTKVEIK (SEQ ID NO: 198).

[0128] In some embodiments, the TAA is EGFR and the second VH chain comprises heavy chain complementarity determining regions 1-3 (HCDRs 1-3) of the VH chain of the 6B3S antibody, comprising the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (sequence number 388).

[0129] In a specific embodiment, the bispecific antibody comprises a combination of a first VH chain comprising HCDRs 1-3, a second VH chain comprising HCDRs 1-3, and a common VL chain comprising LCDRs 1-3, as shown in the table below. [Table 3]

[0130] In addition to binding to ABCG2, the bispecific antibody binds to the TAA listed in the table. The HCDRs in the anti-G2 antibody can be as shown in Table 2. The sequence of the second VH region of the bispecific antibody is shown below. Trastuzumab heavy chain: TIFF0007767322000036.tif51161 The VH region is underlined, and the CDRs are in bold, according to Kabat nomenclature.

[0131] Necitumumab heavy chain: TIFF0007767322000037.tif55163 The VH region is underlined. HCDRs 1 to 3 according to the Kabat nomenclature are shown in bold.

[0132] Atezolizumab heavy chain: The VH region is underlined. HCDRs 1 to 3 according to the Kabat nomenclature are shown in bold.

[0133] 5F9 VH chain: TIFF0007767322000039.tif23163HCDRs 1 to 3 according to Kabat are shown in bold and underlined.

[0134] In some embodiments, a bispecific antibody that binds to both G2 and a TAA may comprise a first VH chain comprising HCDRs 1-3 from an anti-G2 antibody listed in Table 2 (e.g., G2.255), a second VH chain comprising HCDRs 1-3 from an anti-TAA antibody (e.g., anti-CD47 antibody 5F9), and a common VL chain, where the VL chain comprises LCDRs 1-3 from an anti-MDR1 antibody (e.g., MRK16).

[0135] In some embodiments, a bispecific antibody that binds both G2 and a TAA comprises a first VL chain comprising HCDRs 1-3 from an anti-G2 antibody listed in Table 2 and LCDRs 1-3 from an anti-G2 antibody listed in Table 2, where HCDRs 1-3 and LCDRs 1-3 can be from the VH chain and VL chain, respectively, of the same antibody listed in Table 2 or two different antibodies; and a second VH chain comprising HCDRs 1-3 from an anti-TAA antibody, such as the anti-TAA antibodies described herein, and LCDRs 1-3 from an anti-TAA antibody, such as the anti-TAA antibodies described herein, where HCDRs 1-3 and LCDRs 1-3 can be from the same anti-TAA antibody or two different antibodies, respectively, where the first VH chain and first VL chain bind G2 and the second VH chain and second VL chain bind TAA.

[0136] In some embodiments, the subject antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized, or in vitro-generated antibody. As used herein, the term "recombinant" or "modified" antibody is intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell, (ii) antibodies isolated from a recombinant combinatorial antibody library, (iii) antibodies isolated from an animal (e.g., a mouse) transgenic with human immunoglobulin genes, or (iv) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies include humanized, CDR-grafted, chimeric, deimmunized, and in vivo-generated antibodies, and can optionally include constant regions derived from human germline immunoglobulin sequences.

[0137] The modified antibodies may contain modified domains, including where any antibody domain may be modified from its naturally occurring form. In some embodiments, the modified antibodies may contain a modified heavy chain comprising a modified Fc domain, including a modified CH2 and / or CH3 domain. In some cases, the modified Fc domain may utilize an electrostatic steering effect, for example, but not limited to, through the use of the procedure described in Gunasekeran et al. (2010) Journal of Biological Chemistry 285, 19637-19646, the disclosure of which is incorporated herein by reference in its entirety. In some cases, bispecific antibodies are constructed by charge-pair substitutions in the CH3 domain, including, but not limited to, where one heavy chain is modified to contain K392D and K409D substitutions (referred to as "DD") and the other heavy chain is modified to contain E356K and D399K substitutions (referred to as "KK"). Charge-pair substituted chains may preferentially form heterodimers with each other. The numbering of the amino acid substitutions follows the EU numbering system for Ig HC.

[0138] In some cases, the antibodies of the present disclosure comprise charge pair substitutions. In some cases, the antibodies of the present disclosure do not comprise charge pair substitutions. In some cases, alternative means of promoting preferential heterodimer formation of the desired chains can be used.

[0139] In some cases, the modified heavy chain may include a knob-into-hole modification. Knob-into-hole amino acid modification is a rational design strategy in antibody engineering and is used for heavy chain heterodimerization in the production of multispecific antibodies, including bispecific IgG antibodies. For example, when incorporating the knob-into-hole strategy into a bispecific antibody made from two monoclonal antibodies of different specificities, amino acid changes are engineered to create a "knob" in the CH3 of the heavy chain of monoclonal antibody 1 (mAb1) and a "hole" in the CH3 of the heavy chain of monoclonal antibody 2 (mAb2). The knob may be represented by a large amino acid, such as tyrosine (Y), while the hole may be represented by a small amino acid, such as threonine (T). For example, a knob-into-hole modification may create a T22Y substitution in the first CH3 domain and a Y86T substitution in the partner CH3 domain. Examples of knob-into-hole modifications are described in Carter, J. Immunol. Methods, 248(1-2):7-15 (2001); Ridgway, JB et al. Protein Eng. 9(7):617-2 (1996); and Merchant, AM et al. Nat. Biotechnol. 16(7):677-81 (1998), the disclosures of which are incorporated herein in their entireties. Antibodies generated from paired knob-into-hole modified domains generally exhibit a predominant proportion of bispecific heterodimers. The numbering of amino acid substitutions follows the EU numbering system for Ig HC.

[0140] As described above, a subject anti-ABCG2 antibody specifically binds to one or more epitopes of ABCG2. Thus, the epitopes are ABCG2 epitopes. The size of the ABCG2 epitopes bound by an anti-ABCG2 antibody can vary, including when the ABCG2 epitope is formed by a polypeptide having a continuous stretch of the ABCG2 sequence, which can range from 3 aa or less to 12 aa or more, including, but not limited to, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 4 aa-10 aa, 5 aa-10 aa, 6 aa-10 aa, 4 aa-8 aa, 5 aa-8 aa, 6 aa-8 aa, etc.

[0141] In some embodiments, the ABCG2 epitope is, for example, the human ABCG2 sequence: MSSSNVEVFIPVSQGNTNGFPATASNDLKAFTEGAVLSFHNICYRVKLKSGFLPCRKPVE KEILSNINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPSGGLSGDVLINGAPRPANFKCN SGYVVQDDVVMGTLTVRENLQFSAALRLATTMTNHEKNERINRVIQELGLDKVADSKVGT QFIRGVSGGERKRTSIGMELITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTIIF SIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAYNNPADFFLDIING DSTAVALNREEDFKATEIIEPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEKKKK ITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQIIVTVVLGLVIGAIYFGLKNDS TGIQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYISGYYRVSSYFLGKLLSDLLP MRMLPSIIFTCIVYFMLGLKPKADAFFVMMFTLMMVAYSASSMALAIAAGQSVVSVATLL MTICFVFMMIFSGLLVNLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATGN NPCNYATCTGEEYLVKQGIDLSPWGLWKNHVALACMIVIFLTIAYLKLLFLKKYS (SEQ ID NO: 393), or its ECD1 (417-428): KNDSTGIQNRAG (SEQ ID NO: 1), its ECD2 (499-506): LKPKADAF (SEQ ID NO: 2), its ECD3 (557-630): NLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATGNNPCNYATCTGEEYLVKQGIDLSPWGLWKNH (SEQ ID NO: 3), or Mus musculus ABCG2 sequence: MSSSNDHVLVPMSQRNNNGLPRTNSRAVRTLAEGDVLSFHHITYRVKVKSGFLVRKTVEK EILSDINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPKGLSGDVLINGAPQPAHFKCCS GYVVQDDVVMGTLTVRENLQFSAALRLPTTMKNHEKNERINTIIKELGLEKVADSKVGTQ FIRGISGGERKRTSIGMELITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTIIFS IHQPRYSIFKLFDSLTLLASGKLVFHGPAQKALEYFASAGYHCEPYNNPADFFLDVINGD SSAVMLNREEQDNEANKTEEPSKGEKPVIENLSEFYINSAIYGETKAELDQLPGAQEKKG TSAFKEPVYVTSFCHQLRWIARRSFKNLLGNPQASVAQLIVTVILGLIIGAIYFDLKYDA AGMQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYISGYYRVSSYFFGKVMSDLLP MRFLPSVIFTCVLYFMLGLKKTVDAFFIMMFTLIMVAYTASSMALAIATGQSVVSVATLL MTIAFVFMMLFSGLLVNLRTIGPWLSWLQYFSIPRYGFTALQYNEFLGQEFCPGFNVTDN STCVNSYAICTGNEYLINQGIELSPWGLWKNHVALACMIIIFLTIAYLKLLFLKKYS (SEQ ID NO: 394), or its ECD1 (415-428): DLKYDAAGMQNRAG (SEQ ID NO: 395), its ECD2 (499-506): LKKTVDAF (SEQ ID NO: 396), its ECD3 (557-632): NLRTIGPWLSWLQYFSIPRYGFTALQYNEFLGQEFCPGFNVTDNSTCVNSYAICTGNEYLINQGIELSPWGLWKNH (SEQ ID NO: 397), non-human primate sequences, for example, Macaca fascicularis (cynomolgus monkey) sequence: MSSSNVEVFIPMSQENTNGFPTTTSNDRKAFTEGAVLSFHNICYRVKVKSGFLPGRKPVE KEILSNINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPSGGLSGDVLINGALRPTNFKCN SGYVVQDDVVMGTLTVRENLQFSAALRLPTTMTNHEKNERINRVIQELGLDKVADSKVGT QFIRGVSGGERKRTSIGMELITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTIIF SIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAYNNPADFFLDIING DSTAVALNREEDFKATEIIEPSKRDKPLVEKLAEIYVDSSFYKETKAELHQLSGGEKKKK ITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQIIVTVILGLVIGAIYFGLNNDS TGIQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYISGYYRVSSYFFGKLLSDLLP MRMLPSIIFTCIVYFMLGLKPTADAFFIMMFTLMMVAYSASSMALAIAAGQSVVSVATLL MTICFVFMMIFSGLLVNLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATVN NTCNYATCTGEEYLTKQGIDLSPWGLWKNHVALACMIVIFLTIAYLKLLFLKKYS (SEQ ID NO: 398), or its ECD1 (417 to 428): NNDSTGIQNRAG (SEQ ID NO: 399), its ECD2 (499 to 506): LKPTADAF (SEQ ID NO: 400), its ECD3 (557 to 630): NLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATVNNTCNYATCTGEEYLTKQGIDLSPWGLWKNH (SEQ ID NO: 401), or Pan troglodytesThe polypeptide may be formed by a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with a contiguous stretch of ABCG2, including, but not limited to, the ABCG2 sequence: (SEQ ID NO: 402).

[0142] In some embodiments, the ABCG2 epitope can be formed by a mutated ABCG2 polypeptide. The mutated ABCG2 polypeptide can be derived from a human ABCG2 polypeptide. The human ABCG2 polypeptide can contain a mutation that results in an ABCG2 polypeptide having an open conformation. The mutant human ABCG2 polypeptide having an open conformation can contain the substitution: E211Q, numbered with reference to the sequence of the human ABCG2 polypeptide provided herein.In certain embodiments, a mutant human ABCG2 polypeptide having an open conformation may comprise an amino acid sequence that is at least 80% (e.g., at least 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%) identical to the amino acid sequence: (SEQ ID NO: 403).

[0143] In some embodiments, the mutated ABCG2 polypeptide may be derived from a human ABCG2 polypeptide comprising a mutation that results in an ABCG2 polypeptide having a closed conformation. The mutant human ABCG2 polypeptide having a closed conformation may include substitutions, numbered with reference to the sequence of the human ABCG2 polypeptide provided herein: K86M, S87A; K86M, S87A, Q126A; or K86M, S87A, Q126A, R246E. In certain aspects, the mutant human ABCG2 polypeptide having a closed conformation has the amino acid sequence: (SEQ ID NO: 404), (SEQ ID NO: 405), or (SEQ ID NO: 406).

[0144] A subject anti-ABCG2 antibody exhibits high affinity binding to ABCG2. For example, a subject anti-ABCG2 antibody binds human ABCG2 with an affinity of at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, or at least about 10 -12 M or 10-12 The target anti-ABCG2 antibody binds to an epitope present on ABCG2 with an affinity of approximately 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, or about 10 -11 M~about 10 -12 M or 10 -12 It binds with an affinity greater than M.

[0145] A subject anti-ABCG2 antibody exhibits substantially no binding to an epitope formed by amino acids in another protein that is related but has a different sequence (e.g., a related protein with a different sequence, such as EP). Binding of a subject anti-ABCG2 antibody to an epitope formed by amino acids in a protein that is related but has a different sequence is generally nonspecific binding with substantially lower affinity than the specific binding of the anti-ABCG2 antibody to the epitope in ABCG2. A substantially lower affinity is generally at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower affinity.

[0146] A subject anti-ABCG2 antibody can reduce transport of a molecule through an ABCG2 transporter, e.g., human ABCG2. For example, a subject anti-ABCG2 antibody can reduce transport by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the extent of transport in the absence of the anti-ABCG2 antibody.

[0147] In some embodiments, the subject antibodies comprise FR regions that are mammalian sequences, including, for example, rodent, non-human primate, and human sequences (eg, encoded by respective heavy chain FR coding sequences).

[0148] The subject antibodies can comprise a heavy chain variable (VH) region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including 100%) identical to the sequence in the VH region of a VH-VL pair of an antibody shown in Table 2. The subject antibodies can comprise a light chain variable (VL) region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (including 100%) identical to the sequence in the VL region of a VH-VL pair of an antibody shown in Table 2.

[0149] The regions and / or chains of the subject antibodies may or may not be linked by one or more linker regions. If present, the linker region can be from about 5 amino acids to about 50 amino acids in length, e.g., from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa in length.

[0150] Linkers suitable for use with the subject antibodies include "flexible linkers." When present, the linker molecule is generally long enough to allow some degree of flexible movement between the linked regions. Linker molecules are generally about 6-50 atoms in length. Linker molecules can be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules capable of binding to polypeptides can be used in view of the present disclosure.

[0151] Suitable linkers can be easily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0152] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , G.S.G.S.G.S. n (SEQ ID NO: 407), and GGGS n (SEQ ID NO:408), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers are of particular interest because glycine has significantly more access to pi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:409), GGSGG (SEQ ID NO:410), GSGSG (SEQ ID NO:411), GSGGG (SEQ ID NO:412), GGGSG (SEQ ID NO:413), GSSSG (SEQ ID NO:414), and the like. Those skilled in the art will recognize that the design of peptides conjugated to any of the above elements can include linkers that are wholly or partially flexible, whereby the linker can include a flexible linker as well as one or more moieties that impart a less flexible structure.

[0153] In other cases, the flexibility of the hinge region of the antibodies of the present disclosure can be reduced by mutating amino acid C220 to serine or any other natural amino acid, removing C220, removing the entire hinge, or replacing the IgG1 hinge with an IgG3 hinge. Light chains can be connected to form antibodies via their C-terminal cysteines, similar to the situation observed in the human isotype IgA2m. This results in reduced Fab flexibility compared to the Fc and, consequently, reduced cross-linking ability. Another strategy to reduce the flexibility of IgG1 molecules is to replace the IgG1 hinge with an IgG2 hinge or IgG2-like hinge. Alternatively, a variant of the IgG1 hinge similar to the IgG2 hinge can be introduced. This mutant (TH7Δ6-9) contains the mutation T223C and two deletions (K222 and T225), resulting in a short hinge with an additional cysteine.

[0154] Substitution of murine CDRs into a human variable domain framework can result in the retention of their correct spatial orientation; for example, the human variable domain framework adopts the same or a similar conformation as the murine variable domain framework from which the CDRs were derived. This can be achieved by obtaining the human variable domain from a human antibody whose framework sequences show high sequence identity with the murine variable framework domain from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

[0155] Once the complementarity-determining regions of a murine donor immunoglobulin and a suitable human acceptor immunoglobulin have been identified, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with murine amino acid residues should be minimized, as the introduction of murine residues increases the risk of the antibody eliciting a human-anti-murine antibody (HAMA) response in humans. Art-recognized methods for determining immune responses can be performed to monitor HAMA responses in specific patients or during clinical trials. Patients receiving a humanized antibody can undergo immunogenicity assessments at the start of and throughout administration of the therapy. HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from patients using methods known to those skilled in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HAMA response in human subjects.

[0156] Specific amino acids from the human variable region framework residues are selected for substitution based on their potential effect on CDR conformation and / or antigen binding. The non-natural juxtaposition of the murine CDR regions with the human variable framework regions can result in conformational constraints, which, unless corrected by substitution of specific amino acid residues, result in reduced binding affinity.

[0157] The selection of amino acid residues for substitution can be determined, in part, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are generated starting from a solved structure of an immunoglobulin chain or its domain. The chain to be modeled is compared for amino acid sequence similarity with the chain or domain of the solved three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for construction of the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, preferably those sharing at least 60%, 70%, 80%, 90% sequence identity, or more. The solved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain to be modeled and those in the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0158] In some embodiments, the subject antibodies comprise scFv multimers. For example, in some embodiments, the subject antibodies are scFv dimers (e.g., comprising two tandem scFvs (scFv2)), scFv trimers (e.g., comprising three tandem scFvs (scFv3)), scFv tetramers (e.g., comprising four tandem scFvs (scFv4)), or multimers of more than four scFvs (e.g., in tandem). The scFv monomers can be linked via linkers between about 2 amino acids and about 15 amino acids in length, e.g., linkers 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, or 15 aa in length. Suitable linkers include, for example, (Gly) x, (SEQ ID NO: 420), where x is an integer between 2 and 15. Other suitable linkers are discussed above. In some embodiments, each of the scFv monomers in the subject scFv multimer is humanized as described above. In certain aspects, the bispecific antibody can be in any molecular format known in the literature. For example, the bispecific antibody of the present disclosure can have the molecular format described in Spiess C. et al., Mol Immunol. 2015 Oct;67(2 Pt A):95-106.

[0159] In some embodiments, the subject antibodies comprise an immunoglobulin constant region (e.g., Fc region). The Fc region, if present, can be a human Fc region. If a constant region is present, the antibody can comprise both light and heavy chain constant regions. A suitable heavy chain constant region includes CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, and antibodies having any isotype, including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is human isotype IgG1 Fc. The light chain constant region can be lambda or kappa. Antibodies of the invention (e.g., humanized antibodies of the invention) can comprise sequences from more than one class or isotype. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as single chain antibodies in which the heavy and light chain variable domains are linked via a spacer.

[0160] In some embodiments, the subject antibodies comprise a free thiol (—SH) group at the carboxyl terminus, which can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the subject antibody), a scaffold, a carrier, etc.

[0161] The antibody of interest can be covalently attached to a second moiety (e.g., a lipid, a polypeptide other than the antibody of interest, a synthetic polymer, a carbohydrate, a toxin, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks polypeptides through their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and imidoesters crosslink amine-containing polypeptides. At mildly alkaline pH, imidoesters react only with primary amines to form imidoamides, leaving the overall charge of the crosslinked polypeptides unaffected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimidohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-bis[3-(2-pyridyldithio)propionamido]butane (DPDPB).

[0162] Compositions and Formulations The present disclosure provides compositions comprising a subject antibody. The subject antibody compositions can include, in addition to the subject antibody, one or more of the following: a salt, such as NaCl, MgCl, KCl, MgSO, etc.; a buffer, such as Tris buffer, histidine buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a surfactant, such as a non-ionic surfactant such as Tween-20; a protease inhibitor; glycerol, etc.

[0163] The compositions of the present disclosure also include pharmaceutical compositions comprising the antibodies described herein. Generally, the formulation contains an effective amount of the subject antibody. An "effective amount" refers to a dosage sufficient to produce a desired result, such as a reduction in cancer in a subject, a reduction in the growth rate of cancer in a subject, or an improvement in the symptoms of cancer. Generally, the desired result is at least a reduction in the symptoms of cancer, a reduction in the growth of cancer, or a reduction in the size of cancer compared to a control. The subject antibody can be delivered or formulated in a way that avoids the blood-brain barrier.

[0164] In some cases, the antibody may include a delivery enhancer, including where such an enhancer may facilitate crossing the blood-brain barrier, may facilitate increased permeability (e.g., allowing for efficient transdermal delivery), etc.

[0165] In some cases, the antibodies of the present disclosure do not need to be administered in a formulation with a delivery enhancer. In some cases, the antibodies of the present disclosure may themselves enhance permeability across the blood-brain barrier. In some cases, the antibodies of the present disclosure may be used as a delivery enhancer to facilitate crossing of the blood-brain barrier by an anti-tumor agent, such as an immunotherapeutic agent or a chemotherapeutic agent. In some cases, the antibodies of the present disclosure may be used as a delivery enhancer to facilitate crossing of the blood-brain barrier, blood-cerebrospinal fluid (CSF) barrier, blood-testis barrier, or blood-placenta barrier by an active agent, such as another antibody or a chemotherapeutic agent.

[0166] In the subject method, the subject antibody can be administered to a host using any convenient means that can produce the desired therapeutic or diagnostic effect. Thus, the agent can be incorporated into various formulations for therapeutic administration. More specifically, the subject antibody can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0167] In pharmaceutical dosage forms, the subject antibodies can be administered in conjunction with pharmaceutically acceptable excipients, or they can be used alone or in appropriate association and combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0168] The subject antibodies can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, together with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed.

[0169] Pharmaceutical compositions containing the subject antibodies are prepared by mixing the antibody having the desired purity with, optionally, physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, and the like. amino acids such as thiamin, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0170] The pharmaceutical compositions may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, with lyophilized preparations being reconstituted with a sterile solution prior to administration.

[0171] Exemplary antibody concentrations in a subject pharmaceutical composition can range from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0172] Aqueous antibody formulations can be prepared in pH buffer solutions at pHs ranging from about 4.0 to about 7.5, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for pHs within this range include phosphate, histidine, citrate, succinate, acetate, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0173] In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may also be suitable. The term "isotonic" refers to a solution having the same tonicity as some other solution to which it is compared, such as physiological salt solution or serum. Isotonic agents may be used in amounts of about 5 mM to about 350 mM, e.g., 100 mM to 350 nM.

[0174] Surfactants may also be added to antibody formulations to reduce aggregation of the formulated antibody and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Exemplary concentrations of surfactants may range from about 0.001% to about 1% w / v.

[0175] Cryoprotectants may also be added to protect unstable active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Cryoprotectants may be included in amounts of about 10 mM to 500 nM.

[0176] In some embodiments, a subject formulation comprises a subject antibody and one or more of the above-identified agents (e.g., surfactant, buffer, stabilizer, isotonicity agent), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at a concentration ranging from about 0.001 to about 2% (w / v).

[0177] For example, a subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration, and can contain from about 1 mg / mL to about 200 mg / mL of a subject antibody, from about 0.001% to about 1% of at least one surfactant, from about 1 mM to about 100 mM of a buffering agent, optionally from about 10 mM to about 500 mM of a stabilizer, and from about 5 mM to about 305 mM of an isotonic agent, and has a pH of from about 4.0 to about 7.0.

[0178] The subject antibodies can be utilized in aerosol formulations to be administered via inhalation. The subject antibodies can be formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0179] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound of the invention calculated to be sufficient to produce a desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for a subject antibody may depend on the particular antibody employed and the effect to be achieved, as well as the pharmacodynamics associated with each antibody in the host.

[0180] The subject antibodies can be administered as injectable formulations. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid vehicles prior to injection can also be prepared. Preparations can also be emulsified, or the antibody can be encapsulated in liposomal vehicles.

[0181] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods for preparing such dosage forms are known, or will be apparent, to those skilled in the art.

[0182] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0183] In some embodiments, the subject antibodies are formulated in a controlled release formulation. Sustained release preparations can be prepared using methods well known in the art.

[0184] Dosage The appropriate dosage can be determined by the attending physician or other qualified medical professional based on various clinical factors. As is well known in the medical field, the dosage for any single patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, the patient's sex, duration, and route of administration, general health, and other drugs being administered concomitantly. The subject antibodies may be administered in amounts of 1 ng / kg to 20 mg / kg body weight, e.g., 0.1 mg / kg to 10 mg / kg body weight, e.g., 0.5 mg / kg to 5 mg / kg body weight per dose, although doses below or above this exemplary range are contemplated, particularly considering the aforementioned factors. If the regimen is a continuous infusion, it can also range from 1 μg to 10 mg per kilogram of body weight per minute.

[0185] Those of skill in the art will readily appreciate that dosage levels can vary as a function of the particular antibody, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0186] Route of administration The subject antibodies are administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local routes of administration.

[0187] Conventional pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined or adjusted as needed depending on the antibody and / or the desired effect. A subject antibody composition may be administered in a single dose or multiple doses. In some embodiments, a subject antibody composition is administered orally. In some embodiments, a subject antibody composition is administered via the inhalation route. In some embodiments, a subject antibody composition is administered intranasally. In some embodiments, a subject antibody composition is administered topically. In some embodiments, a subject antibody composition is administered intracranially. In some embodiments, a subject antibody composition is administered intravenously.

[0188] The agents can be administered to a host using any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. Generally, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0189] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can be carried out to effect systemic or local delivery of the subject antibody. When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of the pharmaceutical preparation.

[0190] A subject antibody can also be delivered to a subject by enteral administration, which routes of enteral administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

[0191] Treatment refers to at least an amelioration of symptoms associated with a pathological condition afflicting the host, where amelioration is used broadly to refer to at least a decrease in the magnitude of a parameter, e.g., a symptom, associated with the pathological condition being treated, such as cancer and / or cancer growth and associated pain, etc. Thus, treatment also includes situations in which the pathological condition, or at least the symptoms associated therewith, are completely inhibited, e.g., prevented from occurring, or halted, e.g., terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms characterized by the pathological condition.

[0192] A variety of subjects (the term "subject" is used interchangeably herein with the terms "individual" and "patient") can be treated according to the methods of the present disclosure. Generally, such subjects are "mammals" or "mammals," terms used broadly to describe organisms within the class Mammalia, including Carnivora (e.g., dogs and cats), Rodents (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host is a human.

[0193] Kits having unit doses of the subject antibodies, e.g., oral or injectable doses, are provided. In some embodiments, in addition to the container containing the unit dose is an information package insert describing the use of the antibody in treating the pathological condition of interest and the associated benefits.

[0194] nucleic acid The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody of interest, which can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow for expression of the nucleotide sequence in an intended target cell (e.g., a cell that has been genetically engineered to synthesize and / or secrete the encoded antibody).

[0195] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, the light and / or heavy chain immunoglobulin gene promoter and enhancer elements, the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoters, promoters found in the long terminal repeats from retroviruses, the mouse metallothionein-I promoter, and various art-known tissue-specific promoters.

[0196] The nucleotide sequence encoding the antibody of interest can be present in an expression vector and / or a cloning vector. If the antibody of interest comprises two or more separate polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned into the same or separate vectors. Separate polypeptides can be expressed from a single nucleic acid or a single vector using a variety of strategies, including separate promoters, one or more internal ribosome entry sites (IRES), one or more self-cleaving sequences (e.g., 2A cleavage sequences such as P2A, T2A, E2A, and F2A), or combinations thereof. Expression vectors can include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector.

[0197] Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0198] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus), retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0199] For example, nucleic acid as described herein can be introduced into cells in some cases, for example, by contacting cells with nucleic acid.Cells with introduced nucleic acid are generally referred to herein as genetically modified cells.Various nucleic acid delivery methods can be used, including but not limited to, naked nucleic acid delivery, viral delivery, chemical transfection, gene gun, etc.

[0200] cell The present disclosure provides isolated genetically modified cells (e.g., in vitro cells, ex vivo cells, cultured cells, etc.) that have been genetically modified with a nucleic acid of interest. In some embodiments, the isolated genetically modified cells of interest are capable of producing an antibody of interest. In some cases, the genetically modified cells are capable of delivering the antibody, for example, to a subject in need thereof. In some cases, the genetically modified cells can be used for the production, screening, and / or discovery of multispecific antibodies.

[0201] Suitable cells include eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as bacterial cells. Introduction of a nucleic acid of interest into a host cell can be effected, for example, by calcium phosphate precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

[0202] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells, CHO cells, 293 cells, 3T3 cells, Vero cells, Huh-7 cells, BHK cells, PC12 cells, COS cells, COS-7 cells, RAT1 cells, mouse L cells, human embryonic kidney (HEK) cells, HLHepG2 cells, etc.

[0203] In some cases, useful mammalian cells can include cells derived from mammalian tissues or organs. In some cases, the cells used are kidney cells, including, for example, kidney cells from established kidney cell lines such as HEK293T cells.

[0204] In some cases, the cells of the present disclosure may be immune cells. As used herein, the term "immune cells" generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.

[0205] In some cases, useful cells expressing antibodies, such as the multispecific antibodies of the present disclosure, may include producer T cells. Producer T cells engineered to contain nucleic acid sequences encoding the antibodies of the present disclosure may, in some cases, be used to deliver the antibodies to a subject in need thereof.

[0206] In some cases, the immune cells of the present disclosure include immune effector cells comprising a chimeric antigen receptor (CAR) comprising an ABCG2-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the ABCG2-binding domain comprises the heavy chain complementarity-determining region (HCDR) and light chain CDR (LCDR) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. In one embodiment, the intracellular signaling domain can comprise one or more functional signaling domains derived from at least one costimulatory molecule, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. The intracellular signaling domain can comprise a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.

[0207] The immune effector cells can be T cells. The immune effector cells can be autologous cells.

[0208] method As summarized above, the methods of the present disclosure include methods of contacting a cell with an antibody of the present disclosure, methods of treating a subject according to methods that involve administering an antibody of the present disclosure to a subject, and methods of making the elements described in the present application, e.g., antibodies, multispecific antibodies, compositions and formulations, nucleic acids, expression vectors, cells, etc.

[0209] As described above, the methods of the present disclosure include contacting cancer cells with an antibody of the present disclosure to, for example, detect the presence of ABCG2 expression in the cancer cells, measure the expression level of ABCG2 in the cancer cells, or promote and / or enhance cancer cell killing. In some cases, cancer cell killing is mediated by an immune response or immune cells acting on cancer cells bound by the antibody. In some cases, cancer cell killing is mediated by inhibition of cancer cell cytotoxicity, e.g., as a result of antibody-mediated ABCG2 inhibition. In some cases, cancer cell killing is mediated by a combination of inhibition of cancer cell cytotoxicity and an immune-mediated response (e.g., via the Fc region of the antibody). In some cases, the cells contacted with the multispecific antibody may be multidrug-resistant cancer cells. Methods involving contacting cancer cells with an antibody of the present disclosure may or may not include contacting the cancer cells with an additional therapy or active agent, including, for example, a chemotherapeutic agent, an immunotherapeutic agent, a radiotherapeutic agent, etc.

[0210] Contacting cancer cells with a multispecific antibody of the present disclosure generally enhances cancer cell killing, for example, compared to the level of cancer cell killing in the absence of the multispecific antibody. In some cases, when an additional active agent is used, enhanced cancer cell killing may be demonstrated compared to the level of killing observed using the additional active agent alone. The amount of enhanced cancer cell killing resulting from the multispecific antibody varies and may range from at least a 5% increase in cancer cell killing to at least 90% or more, including, but not limited to, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, etc. Such an increase may be compared to contact with one or more additional active agents alone.

[0211] Enhanced killing of cancer cells can be assessed by various means, including, but not limited to, observational tests, in vitro cell-based cytotoxicity assays, flow cytometry, cell viability labeling (e.g., using one or more cell viability stains), and the like.

[0212] Treatment method The present disclosure provides methods of treating cancer, which generally involve administering to an individual in need thereof (e.g., an individual with cancer) an effective amount of an antibody provided herein, alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy). Administration of the antibodies of the present disclosure can be by any convenient, suitable delivery route.

[0213] Thus, administration includes, for example, delivery of an antibody by injection, delivery of an antibody by infusion, delivery of a nucleic acid or expression vector encoding the antibody, delivery of an antibody by administering to a subject cells that express and secrete the antibody, delivery of immune effector cells (e.g., CAR-T cells) that express on their cell surface a chimeric antigen receptor (CAR) comprising an ABCG2-binding domain, a transmembrane domain, and an intracellular signaling domain, where the ABCG2-binding domain comprises the HCDR and LCDR of a pair of VH and VL regions such as those of the antibodies listed in Table 2. Administration of an agent, a nucleic acid encoding an agent, a cell expressing an agent, etc. can include contacting with the agent, contacting with a nucleic acid, contacting with a cell, etc.

[0214] In some embodiments, an effective amount of a subject antibody, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is an amount effective to reduce adverse symptoms of cancer by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the severity of the adverse symptoms in the absence of treatment with the antibody.

[0215] In some embodiments, an effective amount of a subject antibody, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is an amount that is effective to ameliorate cancer (i.e., slow the growth of cancer, stop the growth of cancer, reverse the growth of cancer, kill cancer cells (including tumor cells, etc.)) in the individual being treated. For example, an effective amount of a subject antibody can reduce the rate of cancer growth in an individual or reduce the size of a cancer in an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more, compared to the absence of antibody treatment.

[0216] In some cases, a subject may be treated systemically, including using a subject antibody with or without one or more additional reagents. "Systemic treatment," as used herein, refers to treatment that does not solely target a particular tumor (e.g., a primary tumor or a defined secondary tumor) or a particular cancer-containing tissue (e.g., the liver in the case of liver cancer, the blood in the case of blood cancer, etc.). Systemic treatment is generally directed to the subject's body as a whole and may include, for example, but is not limited to, systemic radiation therapy, systemic chemotherapy, systemic immunotherapy, combinations thereof, etc.

[0217] In some cases, a subject may be treated locally, including using a subject antibody with or without one or more additional reagents. "Local treatment," as used herein, refers to treatment that is specifically directed to the location of the tumor (e.g., the primary tumor or a defined secondary tumor) or to the tissue containing the cancer (e.g., the liver in the case of liver cancer, the blood in the case of blood cancer, etc.). In some cases, a local treatment may also be administered in a manner that affects the environment surrounding the tumor, such as tissue surrounding the tumor, such as tissue immediately adjacent to the tumor. A local treatment generally does not affect or target tissues distant from the cancer site, including the tumor site, such as the primary tumor. Useful local treatments that can be administered in addition to or in combination with a subject antibody include, but are not limited to, surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, etc.

[0218] In some embodiments, a subject treatment method involves administering a subject antibody and one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapy reagents, immunotherapy reagents, other antibodies or multispecific antibody agents, etc. Additional therapies that may be administered to a subject before, during, or after administering a multispecific antibody of the present disclosure to a subject will vary depending on many factors, including, for example, the type of cancer, the subject's previous treatment history, general health, and / or any coexisting conditions. Useful cancer therapies include, but are not limited to, for example, radiation therapy, chemotherapy, immunotherapy, etc.

[0219] Radiation therapy includes, but is not limited to, x-rays or gamma rays delivered from an externally applied source such as a beam or by implantation of small radioactive sources.

[0220] Suitable antibodies for use in cancer therapy include naked antibodies, such as trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), oregovomab (OvaRex™), These include, but are not limited to, lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), and the like, and conjugated antibodies, such as gemtuzumab ozogamicin (Mylortarg™), brentuximab vedotin (Adcetris™), 90Y-labeled ibritumomab tiuxetan (Zevalin™), 131I-labeled tositumomab (Bexxar™), and the like.

[0221] Antibodies suitable for use in cancer therapy also include, but are not limited to, antibodies directed against tumor-associated antigens, such as 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, and TRAILR1. , TRAILR2, RANKL, FAP, tenascin, programmed death-ligand 1 (PD-L1), androgen receptor (AR), Bruton's tyrosine kinase (BTK), BCR-Abl, c-kit, PIK3CA, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEKJ, MEK2, NRAS, RAC1, ESR1, CTLA-4, LAG-3, and TIM-3. These antibodies may be administered as combination therapy with the anti-ABCG2 antibodies provided herein, or as a multispecific antibody comprising at least the antigen-binding portion of one of these antibodies and the antigen-binding portion of the anti-ABCG2 antibodies provided herein.

[0222] Traditional cancer therapies also include targeted therapies against cancer, such as Ado-trastuzumab emtansine (Kadcyla) targeting HER2 (ERBB2 / neu) (approved for use in breast cancer), afatinib (Gilotrif) targeting EGFR (HER1 / ERBB1), HER2 (ERBB2 / neu) (approved for use in non-small cell lung cancer), aldesleukin (Proleukin) targeting renal cell carcinoma, melanoma, and alectinib (Alecensa) targeting ALK. (approved for use in non-small cell lung cancer), alemtuzumab (Campath) targeting CD52 (approved for use in B-cell chronic lymphocytic leukemia), atezolizumab (Tecentriq) targeting PD-L1 (approved for use in urothelial carcinoma and non-small cell lung cancer), avelumab (Bavencio) targeting PD-L1 (approved for use in Merkel cell carcinoma), axitinib (Inlyta) targeting KIT, PDGFRβ, and VEGFR1 / 2 / 3 (approved for use in renal cell carcinoma), and BAF belimumab (Benlysta) targets F (approved for lupus erythematosus), belinostat (Beleodaq) targets HDAC (approved for peripheral T-cell lymphoma), bevacizumab (Avastin) targets VEGF ligands (approved for cervical cancer, colorectal cancer, fallopian tube cancer, glioblastoma, non-small cell lung cancer, ovarian cancer, peritoneal cancer, and renal cell cancer), and blinatumomab (Blincyto) targets CD19 / CD3 (approved for acute lymphoblastic leukemia (precursor B-cell)). ), bortezomib (Velcade) targeting the proteasome (approved for use in multiple myeloma and mantle cell lymphoma), bosutinib (Bosulif) targeting ABL (approved for use in chronic myeloid leukemia), brentuximab vedotin (Adcetris) targeting CD30 (approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma), brigatinib (Alunbrig) targeting ALK (approved for use in non-small cell lung cancer (ALK+)), FLT3, KIT, MET, RET,Cabozantinib (Cabometyx, Cometriq) targets VEGFR2 (approved for use in medullary thyroid cancer and renal cell carcinoma), carfilzomib (Kyprolis) targets the proteasome (approved for use in multiple myeloma), ceritinib (Zykadia) targets ALK (approved for use in non-small cell lung cancer), cetuximab (Erbitux) targets EGFR (HER1 / ERBB1) (approved for use in colorectal cancer and squamous cell carcinoma of the head and neck), and MEK targets Cobimetinib (Cotellic) targets ALK, MET, and ROS1 (approved for use in non-small cell lung cancer), crizotinib (Xalkori) targets BRAF (approved for use in melanoma and non-small cell lung cancer), dabrafenib (Tafinlar) targets BRAF (approved for use in melanoma and non-small cell lung cancer), daratumumab (Darzalex) targets CD38 (approved for use in multiple myeloma), and dasatinib (Sprycel) targets ABL (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia). (approved for use in giant cell tumor of bone), denosumab (Xgeva) targeting RANKL (approved for use in giant cell tumor of bone), dinutuximab (Unituxin) targeting B4GALNT1 (GD2) (approved for use in pediatric neuroblastoma), durvalumab (Imfinzi) targeting PD-L1 (approved for use in urothelial carcinoma), elotuzumab (Empliciti) targeting SLAMF7 (CS1 / CD319 / CRACC) (approved for use in multiple myeloma), and enasideri targeting IDH2. Idhifa (approved for use in acute myeloid leukemia), erlotinib (Tarceva) which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer and pancreatic cancer), everolimus (Afinitor) which targets mTOR (approved for use in neuroendocrine tumors of pancreatic, gastrointestinal, or pulmonary origin, renal cell carcinoma, unresectable subependymal giant cell astrocytoma, and breast cancer), and gefitinib (Iressa) which targets EGFR (HER1 / ERBB1) (approved for use in non-small cell lung cancer).Ibritumomab tiuxetan (Zevalin) targets CD20 (approved for use in non-Hodgkin's lymphoma), ibrutinib (Imbruvica) targets BTK (approved for use in mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenström's hypergammaglobulinemia), idelalisib (Zydelig) targets PI3Kδ (approved for use in chronic lymphocytic leukemia, follicular B-cell non-Hodgkin's lymphoma, and small lymphocytic lymphoma), and imatinib (Gleevec) targets KIT, PDGFR, and ABL. Approved for use in GI stromal tumors (Kit+), dermatofibrosarcoma protuberans, and multiple hematologic malignancies; ipilimumab (Yervoy) targeting CTLA-4 (approved for use in melanoma); ixazomib (Ninlaro) targeting the proteasome (approved for use in multiple myeloma); lapatinib (Tykerb) targeting HER2 (ERBB2 / neu) and EGFR (HER1 / ERBB1) (approved for use in breast cancer (HER2+)); and lenvatinib (Lenvima) targeting VEGFR2 (renal cell carcinoma). , approved for use in thyroid cancer), midostaurin (Rydapt) targeting FLT3 (approved for use in acute myeloid leukemia (FLT3+)), necitumumab (Portrazza) targeting EGFR (HER1 / ERBB1) (approved for use in squamous non-small cell lung cancer), neratinib (Nerlynx) targeting HER2 (ERBB2 / neu) (approved for use in breast cancer), nilotinib (Tasigna) targeting ABL (approved for use in chronic myeloid leukemia), and PARP targeting Niraparib (Zejula) (approved for use in ovarian cancer, fallopian tube cancer, and peritoneal cancer), nivolumab (Opdivo) which targets PD-1 (approved for use in colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, melanoma, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma), obinutuzumab (Gazyva) which targets CD20 (approved for use in chronic lymphocytic leukemia and follicular lymphoma), and ofatumumab (Arzerra, HuMax-CD20) which targets CD20 (approved for use in chronic lymphocytic leukemia),Olaparib (Lynparza) targets PARP (approved for ovarian cancer), olaratumab (Lartruvo) targets PDGFRα (approved for soft tissue sarcoma), osimertinib (Tagrisso) targets EGFR (approved for non-small cell lung cancer), palbociclib (Ibrance) targets CDK4 and CDK6 (approved for breast cancer), and panitumumab (Vectibix) targets EGFR (HER1 / ERBB1). panobinostat (Farydak), which targets HDAC (approved for use in multiple myeloma); pazopanib (Votrient), which targets VEGFR, PDGFR, and KIT (approved for use in renal cell carcinoma); pembrolizumab (Keytruda), which targets PD-1 (approved for use in classical Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+), squamous cell carcinoma of the head and neck, and solid tumors (MSI-H)); and HER2 (ERBB2 / neu) pertuzumab (Perjeta) targets ABL (approved for use in breast cancer (HER2+)); ponatinib (Iclusig) targets ABL, FGFR1-3, FLT3, and VEGFR2 (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia); ramucirumab (Cyramza) targets VEGFR2 (approved for use in colorectal cancer, gastric cancer or esophagogastric junction (GEJ) adenocarcinoma, and non-small cell lung cancer); and ramucirumab targets KIT, PDGFRβ, RAF, RET, and VEGFR1 / 2 / 3. regorafenib (Stivarga) (approved for use in colorectal cancer, gastrointestinal stromal tumors, and hepatocellular carcinoma), ribociclib (Kisqali) (approved for use in breast cancer (HR+, HER2-)) targeting CDK4 and CDK6, rituximab (Rituxan, Mabthera) (approved for use in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis), and rituximab / hyaluronidase human (Rituxan Hycela) (approved for use in chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma) targeting CD20.Romidepsin (Istodax) targets HDAC (approved for cutaneous T-cell lymphoma and peripheral T-cell lymphoma), rucaparib (Rubraca) targets PARP (approved for ovarian cancer), ruxolitinib (Jakafi) targets JAK1 / 2 (approved for myelofibrosis), siltuximab (Sylvant) targets IL-6 (approved for multicentric Castleman disease), and sipuleucel-T (Provenge) targets prostate cancer. approved for use in basal cell carcinoma), sonidegib (Odomzo) targets smooth muscle (approved for use in basal cell carcinoma), sorafenib (Nexavar) targets VEGFR, PDGFR, KIT, and RAF (approved for use in hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer), temsirolimus (Torisel) targets mTOR (approved for use in renal cell carcinoma), tositumomab (Bexxar) targets CD20 (approved for use in non-Hodgkin's lymphoma), and thrombus targeting MEK. Lametinib (Mekinist) (approved for use in melanoma and non-small cell lung cancer), trastuzumab (Herceptin) targeting HER2 (ERBB2 / neu) (approved for use in breast cancer (HER2+) and gastric cancer (HER2+)), vandetanib (Caprelsa) targeting EGFR (HER1 / ERBB1), RET, and VEGFR2 (approved for use in medullary thyroid cancer), and vemurafenib (Zelboraf) targeting BRAF (approved for use in melanoma). These antibodies include, but are not limited to, venetoclax (Venclexta) targeting BCL2 (approved for use in chronic lymphocytic leukemia), vismodegib (Erivedge) targeting PTCH and smooth muscle (approved for use in basal cell carcinoma), vorinostat (Zolinza) targeting HDAC (approved for use in cutaneous T-cell lymphoma), and Ziv-aflibercept (Zaltrap) targeting PIGF and VEGFA / B (approved for use in colorectal cancer). These antibodies may be administered in combination with the anti-ABCG2 antibodies provided herein.

[0223] Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity, (2) inhibitors of serine / threonine kinase activity, (3) tumor-associated antigen antagonists such as antibodies that specifically bind to tumor antigens, (4) apoptosis receptor agonists, (5) interleukin-2, (6) interferon-α, (7) interferon-γ, (8) colony-stimulating factors, (9) inhibitors of angiogenesis, and (10) antagonists of tumor necrosis factor.

[0224] Chemotherapeutic or antitumor agents are non-peptide (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents (e.g., nitrosoureas), antimetabolites (e.g., methotrexate), antitumor antibiotics (e.g., anthracyclines), plant alkaloids (e.g., vinca alkaloids, taxanes, etc.), toposiomerase inhibitors, and steroid hormones.

[0225] Drugs that act to reduce cell proliferation are known in the art and are widely used. Such drugs include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkylsulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0226] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0227] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics, such as anthracyclines , daunorubicin hydrochloride (daunomycin, rubidomycin, cerubicin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives; phenoxyzolidinic cyclopeptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrroloindoleziones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like.

[0228] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0229] Microtubule-affecting agents with antiproliferative activity are also suitable for use, including, but not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterol, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones (including, but not limited to, etopirone A, epothilone B, discodermolide), estramustine, and nocodazole.

[0230] Hormonal modulating agents and steroids (including synthetic analogs) suitable for use include, but are not limited to, adrenocorticosteroids such as prednisone, dexamethasone, and the like; estrogens and pregestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; adrenocortical suppressants such as aminoglutethimide, 17α-ethinylestradiol, diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testolone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex. Estrogen stimulates proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0231] Other chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; ureas such as hydroxyurea; and hydrazines such as N-methylhydrazine; epidophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, and the like. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azasporin, leflunomide, mizoribine, azaspirane (SKF105685); Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline, and the like.

[0232] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be prepared by techniques known to those skilled in the art (WO 94 / 07882, WO 94 / 0788 1, WO94 / 07880, WO94 / 07876, WO93 / 23555, WO93 / 10076, U.S. Pat. Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP 590,267), or can be obtained from a variety of commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis).

[0233] Paclitaxel should be understood to refer not only to the common chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., the above-mentioned Taxotere™ docetaxel) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, or protein-bound paclitaxel such as Abraxane®).

[0234] The term "taxane" also encompasses various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113, the piperazino and other derivatives described in WO 99 / 14209, the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680, the 6-thio derivatives described in WO 98 / 28288, the sulfenamide derivatives described in U.S. Patent No. 5,821,263, and the taxol derivatives described in U.S. Patent No. 5,415,869. Prodrugs of paclitaxel are further included, including, but not limited to, those described in WO 98 / 58927, WO 98 / 13059, and U.S. Patent No. 5,824,701.

[0235] Useful immunotherapies include anti-PD-1 / PD-L1 immunotherapies and / or other immunotherapies that target immune checkpoint markers such as, for example, CTLA-4, LAG-3, and TIM-3, which can be targeted in a therapeutic method. Anti-PD-1 / PD-L1 immunotherapy, for example, includes, but is not limited to, administering to a subject an effective amount of one or more anti-PD-1 / PD-L1 therapeutic antagonists, including, but not limited to, OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), Tecentriq™ (atezolizumab), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BMS-242, etc. These antibodies may be administered as combination therapy with the anti-ABCG2 antibodies provided herein.

[0236] CTLA-4, also known as CD152, binds to CD80 and CD86. Antibodies against CTLA-4 have been approved for treating several cancer types. The co-inhibitory effect of CTLA-4 with other immunotherapies makes CTLA-4 an excellent candidate for use in combination with other immunotherapies to treat certain cancers. TIM-3 can also be targeted for immunotherapy in some cancer types.

[0237] LAG-3 is currently undergoing clinical trials for the treatment of cancer. Anti-LAG-3 immunotherapy includes the use of antagonist LAG-3 antibodies that can both activate T effector cells (by downregulating LAG-3 inhibitory signals in preactivated LAG-3+ cells) and inhibit induced (i.e., antigen-specific) Treg suppressive activity. Useful LAG-3 antagonist antibodies include leratolimab (BMS-986016, developed by Bristol-Myers Squibb), IMP701 (developed by Immutep), and TSR-033 (anti-LAG-3 mAb, developed by TESARO, Inc.).

[0238] Immunotherapy also includes T cell-based immunotherapies, such as adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) T cell therapy. For example, a subject may be administered a population of CAR T cells engineered to target an antigen expressed by the subject's cancer. T cell-based therapy, in some cases, may involve obtaining a cell sample, such as a blood sample or tumor biopsy, from the subject and culturing immune cells from the sample ex vivo, with or without genetic modification of the cultured immune cells. For example, immune cells may be obtained from the subject, cultured ex vivo, and engineered with a CAR specific to an antigen expressed by the cancer to produce a population of CAR T cells. The CAR T cells may then be reintroduced into the subject to target the cancer. T cell-based immunotherapy may be configured in various ways, such as by targeting different antigens or by collecting / culturing different cell types, depending on the particular cancer being treated. In addition, T cell-based immunotherapies can be administered systemically, e.g., by intravenous injection, or locally, e.g., by infusion (e.g., intraperitoneal infusion, pleural catheter infusion, etc.), direct injection, etc.

[0239] In some cases, the therapeutic methods described herein may include administering to a subject one or more inhibitors of multidrug resistance transporters, including, but not limited to, multidrug resistance transporters other than ABCG2. Useful inhibitors of multidrug resistance transporters include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and small molecule inhibitors. Inhibitors of multidrug resistance transporters include ABC transporter inhibitors.

[0240] Individuals suitable for treatment using the methods of the present disclosure include individuals who have cancer, individuals who have been diagnosed with cancer, individuals who are being treated for cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc., individuals who have been treated for cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) but have not responded to treatment, and individuals who have been treated for cancer (e.g., with one or more of chemotherapy, radiation therapy, antibody therapy, surgery, etc.) and initially responded to treatment but then relapsed, i.e., their cancer has recurred.

[0241] The methods of the present disclosure can be used to target and treat a variety of cancers, including, for example, primary cancers, secondary cancers, regrowing cancers, recurrent cancers, refractory cancers, etc. For example, in some cases, the methods of the present disclosure can be used as an initial treatment for a primary cancer identified in a subject. In some cases, the methods of the present disclosure can be used as a non-primary (e.g., second-line or later) treatment, for example, in subjects with cancer that is refractory to a previous treatment, in subjects with cancer that is regrowing after a previous treatment, in subjects with a mixed response to a previous treatment (e.g., a positive response to at least one tumor in the subject and a negative or neutral response to at least one second tumor in the subject), etc.

[0242] In some cases, the methods of the present disclosure can be used to treat subjects with drug-resistant cancers, such as multidrug-resistant cancers. Multidrug resistance (MDR) is the mechanism by which many cancers develop resistance to chemotherapeutic agents, resulting in minimal cell death and the expansion of drug-resistant tumors. MDR cancers can involve one or more resistance mechanisms, including, but not limited to, increased expression of efflux pumps, reduced absorption of drugs, inhibition of cell death or apoptosis, modulation of drug metabolism, etc. In some cases, the methods of the present disclosure can prevent, reverse, or circumvent MDR.

[0243] In some cases, the methods of the present disclosure can include treating a subject having a cancer that is resistant to a first agent with an effective amount of a subject antibody described herein in combination with a second agent that is different from the first agent. For example, in some cases, the subject's cancer can be resistant to a first chemotherapeutic agent, and the subject can be treated by administering an effective amount of a subject antibody described herein in combination with a second chemotherapeutic agent that is different from the first chemotherapeutic agent. Various combinations of first and second chemotherapeutic agents can be used, depending, for example, on the type of cancer being treated, the likelihood of developing resistance, etc.

[0244] Many cancers are known to develop drug resistance. For this and other reasons, the methods of the present disclosure may find use in a variety of cancers, including, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendix cancer, astrocytoma, atypical teratogenic / rhabdoid tumor, basal cell carcinoma, bile duct cancer (extrahepatic), bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, and malignant fibrohistiocytoma, etc.), brain stem glioma, brain tumors (e.g., astrocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharynx tumor, etc.), and the like. Cervix, epithelioma, etc.), breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumor, Burkitt's lymphoma, carcinoid tumor (e.g., pediatric, gastrointestinal, etc.), tumor of unknown primary, cardiac (heart) tumor, central nervous system (e.g., atypical teratogenic tumor / rhabdoid tumor, embryonal tumor, germ cell tumor, lymphoma, etc.), cervical cancer, pediatric cancer, spinal cord tumor, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, duct (e.g., bile duct, extrahepatic, etc.), non-invasive breast duct (DCIS), germinal tumor, endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), fibrous histiocytoma of bone (e.g., malignant osteosarcoma, etc.), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, pancreatic cancer, hepatocellular (liver) cancer , histiocytosis (e.g., Langerhans cell, etc.), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (e.g., pancreatic neuroendocrine tumors, etc.), Kaposi's sarcoma, kidney cancer (e.g., renal cell, Wilms' tumor, childhood kidney tumors, etc.), Langerhans cell histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), hairy cell, etc.), lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.),Lymphoma (e.g., AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system (CNS), etc.), macroglobulinemia (e.g., Waldenstrom, etc.), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary, midline duct carcinoma with Nat gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplasia / bone Myeloproliferative neoplasms, myeloid leukemia (e.g., chronic (CML)), myeloid leukemia (e.g., acute (AML)), myeloproliferative neoplasms (e.g., chronic tumors), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cancer (e.g., lip), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer (e.g., epithelial, germ cell tumor, low-grade malignant tumor), pancreatic cancer, pancreatic neuroendocrine tumor (islet cell tumor), papillary tumor, Paranasal sinus tumors, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell carcinoma of the renal pelvis and urethra, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, tumors (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 These include, but are not limited to, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer (e.g., with unknown primary site, metastatic, etc.), stomach (gastric) cancer, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureter and renal pelvis cancer, urethral cancer, uterine cancer (e.g., endometrial cancer, etc.), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's hypergammaglobulinemia, Wilms' tumor, etc.

[0245] In some cases, the treatment methods described herein can be performed on subjects who have previously undergone one or more conventional treatments.For example, in the case of oncology, in some cases, the methods described herein can be performed following conventional cancer treatments, including but not limited to conventional chemotherapy, conventional radiation therapy, conventional immunotherapy, surgery, etc. In some cases, the methods described herein can be used when the subject is not responding to conventional therapy or is refractory to conventional therapy.In some cases, the methods described herein can be used when the subject is responding to conventional therapy.

[0246] In some cases, the methods of the present disclosure can be used to target, treat, or eliminate subjects with minimal residual disease (MRD) remaining after a previous cancer treatment. Targeting, treating, and / or eliminating MRD can be pursued using the methods regardless of whether the MRD is refractory to a previous treatment. In some cases, the methods of the present disclosure can be used to target, treat, and / or eliminate subjects with MRD after a determination that the MRD is refractory to a previous treatment or to one or more available treatment options other than treatment with a multispecific antibody described herein.

[0247] In some cases, the methods can be used prophylactically for surveillance. For example, a subject in need thereof can be administered treatment with one or more of the mono- or multispecific antibodies described herein when the subject does not have detectable disease but is at risk of developing a recurrent cancer, including, for example, a drug-resistant cancer. In some cases, a prophylactic approach can be used when the subject is at particularly high risk of developing a primary cancer that is predicted to be or become drug-resistant. In some cases, a prophylactic approach can be used when the subject has previously been treated for cancer and is at risk of recurrence or developing drug resistance.

[0248] In some cases, the methods of the present disclosure may involve analyzing the cancer for expression of one or more markers or therapeutic targets. For example, in some cases, the methods may involve analyzing a cancer sample from a subject to determine whether the cancer expresses ABCG2 above a predetermined threshold, a cancer-associated antigen or MDR-1 above a predetermined threshold, or both ABCG2 and a TAA, or both ABCG2 and MDR1 above a predetermined threshold.

[0249] In some cases, whether a subject is treated with an antibody of the present disclosure may depend on the results of the ABCG2 expression assessment, the cancer-associated antigen expression, or both. For example, in some cases, if the cancer expresses ABCG2 at or above a predetermined threshold, the subject may be treated with an anti-ABCG2 antibody of the present disclosure or a multispecific antibody of the present disclosure; if the cancer expresses ABCG2 below the predetermined threshold, the subject may be treated with a multispecific antibody of the present disclosure without being treated with an anti-ABCG2 antibody.

[0250] Any convenient assay can be used to analyze ABCG2 and / or cancer-associated antigen levels, including, but not limited to, flow cytometry, nucleic acid-based assays (e.g., amplification, sequencing, etc.), cell cytometry, immunohistochemistry, etc. Any convenient biological sample can be used, including, but not limited to, a cancer biopsy sample. A useful predetermined threshold for assessing the expression of one or more markers and / or targets can be determined by any convenient and appropriate method, including comparing the measured expression levels with a corresponding control. For example, in some cases, a useful predetermined threshold for the level of ABCG2 and / or cancer-associated antigen assayed in a sample can correspond to the level of ABCG2 and / or cancer-associated antigen measured in reference cells, such as healthy / normal cells.

[0251] How to make it As summarized above, the methods of the present disclosure also include methods for making and / or identifying the antibodies described herein. The subject antibodies can be produced by any known method, for example, conventional synthetic methods for protein synthesis, recombinant DNA methods, etc.

[0252] If the subject antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When a polypeptide is chemically synthesized, synthesis can proceed via liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is bound to an insoluble support, followed by sequential addition of the remaining amino acids to the sequence, is an example of a suitable method for chemically synthesizing the subject antibody. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing the subject antibody.

[0253] Standard recombinant methods can be used to produce the subject antibodies. For example, nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. The expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences, and the collection and purification of the antibody.

[0254] Due to the degeneracy of the genetic code, a variety of nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be generated by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of an earlier prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for preparing substitution, deletion, and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, target polypeptide DNA is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template incorporating the oligonucleotide primer, encoding the selected modification within the target polypeptide DNA.

[0255] Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selectable markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline-resistance, kanamycin-resistance, or neomycin-resistance) to permit detection of those cells transformed with the desired DNA sequences.

[0256] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding an antibody of interest. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0257] Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, along with suitable vectors having expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization, among others.

[0258] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce polypeptides of the invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, HEK cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0259] Once synthesized (either chemically or recombinantly), whole antibodies, dimers thereof, individual light and heavy chains, or other forms of the subject antibodies (e.g., scFv, etc.) can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (See generally, Scopes, Protein Purification, Springer-Verlag, NY, (1982)). The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% or more pure, and free of contaminants such as, for example, cellular debris, macromolecules other than the subject antibody, etc.

[0260] In some embodiments, methods for generating multispecific antibodies of the present disclosure may include producing candidate antibodies and screening for activity. Such methods may generate multispecific antibodies that specifically bind to cells that express both ABCG2 and a cancer-associated antigen or MDR1 by using a series of steps. The steps of such methods may include producing a multispecific antibody or antibodies each comprising or expected to comprise an ABCG2-binding domain and a cancer-associated antigen-binding domain / MDR1-binding domain; contacting a first test cell expressing ABCG2 and the cancer-associated antigen or MDR-1 with the multispecific antibody or antibodies; contacting a second cell expressing either ABCG2 or the cancer-associated antigen / MDR1 with the multispecific antibody or antibodies; comparing binding of the multispecific antibody to the first cell with binding of the multispecific antibody to the second cell to determine a binding specificity ratio; and identifying the multispecific antibody or one or more of the antibodies as specific for cells expressing both ABCG2 and the cancer-associated antigen / MDR1 when the ratio exceeds a predetermined threshold. When such a threshold for comparative binding is used, the threshold may vary and may range from 1.5:1 or greater, including, but not limited to, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1, etc.

[0261] Various cells can be used in such methods, including, but not limited to, the cells described herein. In some cases, antibody binding can be performed on both cells that express only ABCG2 and cells that express only a cancer-associated antigen. For example, in some cases, the method may further include, in relation to the above steps, contacting a second cell that expresses ABCG2 but not a cancer-associated antigen with a multispecific antibody, whereas the method may further include contacting a third cell that expresses a cancer-associated antigen but not ABCG2 with the multispecific antibody.

[0262] In some cases, such methods may use one or more controls, including, but not limited to, control cells, control reagents, etc. Useful control cells include those with known expression or lack of known expression of one or more relevant genes or proteins. Useful control reagents may include, but are not limited to, control antibodies, such as monospecific antibodies against known targets. For example, in some cases, such methods of the present disclosure may further include contacting the first cell, the second cell, and / or the third cell with a control antibody selected from a monospecific anti-ABCG2 antibody and a monospecific anti-cancer-associated antigen antibody. Depending on the particular method used, various other or additional controls may be used as appropriate, as needed.

[0263] kit Aspects of the present disclosure also include kits. Kits may include, for example, any combination of antibodies, multispecific antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. described herein. A subject kit may include one or more of a subject antibody, a nucleic acid encoding the same, or cells containing a subject nucleic acid. Kits may be configured for a variety of purposes, including, for example, therapeutic kits (e.g., the kit may include an anti-ABCG2 antibody or multispecific antibody and one or more additional active agents, such as, for example, a chemotherapeutic agent), kits for producing antibodies, kits for screening antibodies, etc.

[0264] Optional components of the kits may vary and include, for example, buffers, protease inhibitors, etc. Where the subject kits include a subject nucleic acid, the nucleic acid may also have restriction sites, multiple cloning sites, primer sites, etc. The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined in a single container, if desired.

[0265] In addition to the above components, the subject kits can include instructions for using the kit components to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present on the label of the container of the kit or its components (i.e., associated with the packaging or subpackaging) as a package insert. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, such as a compact disc read-only memory (CD-ROM), digital versatile disc (DVD), diskette, etc. In still other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the Internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0266] Exemplary Non-Limiting Aspects of the Disclosure The above-described aspects of the present subject matter, including embodiments, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure are provided below. As will be apparent to one skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. It will be apparent to one skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the present invention.

[0267] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0268] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are the experiments below intended to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0269] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Reagents, cloning vectors, cells, and kits for the methods mentioned in or related to this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and repositories such as, for example, Addgene, Inc., American Type Culture Collection (ATCC).

[0270] Example 1: Generation of antibodies that specifically bind to cells expressing ABCG2 Materials and Methods antibody generation Wild-type (WT) human ABCG2 and mutant ABCG2 were used for immunization. Various mutants of ABCG2 were generated that constrain the pump in either the open or closed conformation. The mutations in human and Macaca fascicularis ABCG2 used to generate ABCG2 mutants constrained in the open conformation are E221Q. The mutations in human ABCG2 used to generate ABCG2 mutants constrained in the closed conformation are (i) K86M, S87A, (ii) K86M, S87A, Q126A, and / or (iii) K86M, S87A, Q126A, R246E. 3T3-ABCG2 and C6-ABCG2-expressing stable cells were generated by transfecting 3T3 and C6 cells with the pMono-hygro-ABCG2 plasmid followed by selection with 0.2 mg / ml or 0.5 mg / ml hygromycin. Virus-like particles displaying AGCG2 protein were generated by cotransfection of 293T cells with a plasmid carrying the AGCG2 gene and retroviral Gag protein, as previously described (Popova et al. 2008, J Virol 82(3):1389-1398).

[0271] Female mice or rats were immunized for 8–12 weeks with ABCG2 (wild-type or mutant) human or cynomolgus monkey DNA and / or ABCG2 (wild-type or mutant)-expressing cells or virus-like particles using various prime-boost strategies. In some cases, more than one antigen (one or more of wild-type or mutant ABCG2) was used for immunization to increase the diversity of antibodies generated and enhance the immune response. Spleen and lymph node cells from vaccinated animals were fused with SP2 / 0 myeloma cells (hybridoma technology). Hybridoma supernatants were screened for the presence of anti-ABCG2 antibodies by flow cytometry and then screened for functional activity in cell killing (chemosensitization) and efflux inhibition assays. CDRs from selected murine IgGs were cloned into a mammalian IgG1 backbone expression vector for full-length IgG1 antibody expression and production in HEK293 host cells via transfection using standard protocols, as described below.

[0272] Expression vector To generate antibody expression vectors, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with either the human IgG1 constant heavy chain or the human IgG1 kappa constant light chain, respectively, which had been previously inserted into a common recipient expression vector optimized for expression in mammalian cell lines. The genes to be expressed were cloned into the pCI-neo mammalian expression vector (Promega), which uses the full-length human cytomegalovirus (CMV) immediate-early promoter for high-level gene expression. The two antibody chains were cloned into two different vectors.

[0273] The N-terminal signal sequences from mouse IgG heavy and kappa light chains were used for secretory expression of the heavy and light chains, respectively. The signal peptides were cleaved during expression, leaving intact N-termini. In the Fab constructs, the C-terminus of the CH1 IgG1 constant region was fused to a 6xHis tag for purification.

[0274] mAb production Polymer-based co-transfection of Expi293 cells (A14527, ThermoFisher) cells grown in suspension with mammalian expression vectors was used according to the manufacturer's recommendations to express antibody constructs.

[0275] Approximately 6 days after transfection, cells were harvested by centrifugation. Specifically, 1 μg of total coding DNA per ml of culture to be transfected was diluted in Opti-MEM® medium (Life Technologies) and incubated with Expifectamine reagent (Life Technologies) in the same medium for 20 minutes. The mixture was then added to Expi293® cells growing in suspension in Expi293® Expression Medium (Life Technologies) at 2.5 million cells / ml at 37°C in air with 8% CO2. After 6 days, the medium containing the antibody construct was harvested by centrifugation.

[0276] mAb purification To purify antibody formats containing human and mouse Fc, 10 μl of MabSelect™ SuRe™ (GE Healthcare) per ml of supernatant was added to the collected medium and stirred overnight at 4°C. The next day, Protein A resin was applied in a 24-well filter plate using a vacuum manifold unit (Pall Lifesciences, USA). The resin was washed with PBS, and the antibody was eluted with 50 mM phosphate pH 3 and neutralized with 10x PBS pH 13.

[0277] Analytical Tests for mAb (GXII Reduced and Non-Reduced) The purity and monomer content of the final protein preparation were determined by high-throughput analysis on a Caliper LabChip GXII using the Protein Express LabChip Kit (Perkin-Elmer) according to the manufacturer's instructions. The chip was automatically primed on the instrument with a polymer solution containing 0.2% SDS and a fluorescent dye. The destaining channel was filled with a polymer solution without SDS or dye. Briefly, proteins in reduced and nonreduced conditions were prepared by mixing small sample volumes (2–5 μL) with Caliper sample buffer, with or without DDT. Samples were denatured at 75°C for 5 min and centrifuged at 2000 g for 3 min before electrophoresis. Electropherograms were generated using LabChip GXII Touch software (Perkin-Elmer).

[0278] Analytical Testing for mAb (HPLC) The purity and monomer content of the final protein preparation were determined by high-throughput HPLC analysis. Size-exclusion chromatography (SEC) was performed on an Infinity 1260 Agilent HPLC system using an Advancebio SEC 300A 4.6 x 300 mm, 2.7 μm (p / n PL1580-5301) (Agilent Technologies). Injections were performed under isocratic elution conditions using a mobile phase of PBS, 400 mM sodium chloride, pH 7.4, and detection was by absorbance at 280 nm. Quantification was based on the relative areas of the detected peaks.

[0279] The subject antibodies can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% or more pure, and are free of contaminants such as, for example, cellular debris, macromolecules other than the subject antibodies, etc.

[0280] Transient transfection of 293T cells 293T cells were transiently transfected with a human P-glycoprotein-tagged ORF clone in the pLenti-C-Myc-DDK-P2A-Puro plasmid using an optimized PEIPro™ transfection protocol (Polyplus). DNA and JetPEI® were diluted in culture medium and then gently mixed for approximately 10 minutes. This mixing resulted in the formation of a transfection complex, which was added directly to the cell culture. Efflux blockade was measured using a Multidrug Resistance Direct Dye Efflux Assay (Chemicon) according to the manufacturer's protocol.

[0281] Emissions interception experiment procedure 293T_ABCG2_OX cells were washed several times and seeded into 96-well plates in 50 μl aliquots per well at a cell density of 2 × 10e6 cells per ml in phenol red-free DMEM. Cells were mixed with 50 μl of antibody or small molecule inhibitor of ABCG2—fumitremordin C (FTC) or its tetracyclic analog KO143—at a final concentration of 1 μM. Cells were then incubated for 1 hour at 37°C in the presence of 2.5 μM mitoxantrone. Cells were then washed twice and finally resuspended in 200 ml of PBS. Mitoxantrone fluorescence was measured using an excitation of 635 nM and an emission of 647 nM. Protocols for measuring efflux blockade can be found in Szabo E, et al., PLoS One. 2018 Jan 17;13(1):e0190629; Deeken JF, et al., Mol Pharmacol. 2009 Nov;76(5):946-56).

[0282] Titer measurement of monoclonal antibodies that bind to KPG2 Binding titration of recombinant antibodies to KPG2 transfectants was performed by serial dilution of the antibody starting at approximately 666 nM. Antibodies diluted in flow cytometry buffer were incubated with cells on ice for 30 minutes. After two washes with flow cytometry buffer, bound antibodies were detected with PE-labeled F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) diluted 1:200 in flow cytometry buffer and incubated with cells on ice for 20 minutes. After two washes with flow cytometry buffer, fluorescence was measured using an Attune NxT flow cytometer. Data were analyzed using GraphPad Prism 8.0 software to determine EC50 values.

[0283] The bispecific antibodies were characterized by binding titration and chemotoxicity assays.

[0284] Cell binding assay. Antibody binding to cells was assessed by flow cytometry. 293T cells stably transfected to express human or cynomolgus monkey ABCG2 (293T_ABCG2_OX) were washed once with flow cytometry buffer (PBS + 2% FBS + 0.02% sodium azide), resuspended in flow cytometry buffer at 2 × 10^6 cells / mL, and dispensed into a 96-well microtiter plate at 0.1 mL / well. Recombinant antibodies were added to cells at 5 μg / mL for initial binding confirmation or serially diluted in flow cytometry buffer starting from 100 μg / mL. After incubating cells on ice for 30 minutes, the cells were washed twice with flow cytometry buffer. Bound antibodies were detected with PE-labeled F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) and assessed using an Attune NxT flow cytometer. The EC50 is calculated to be the concentration of antibody that produces half the maximal response.

[0285] Cytotoxicity assay. The effect of antibodies on topotecan cytotoxicity was evaluated in 293T_ABCG2_OX cells, 293T cells stably transfected to express ABCG2. Cells were seeded into white, flat-bottom, 96-well tissue culture plates at 5000 cells / well in 0.05 mL of assay medium (DMEM + 10% FBS). Topotecan was prepared at 2× final assay concentration by serial dilution from 200 μM into assay medium containing 100 μg / mL (2× final concentration) of test or control antibody or 20 μM (2× final concentration) of the small molecule ABCG2 inhibitor fumitremorgin C. An equal volume (0.05 mL) of the topotecan / antibody mixture was added to the 293T_ABCG2_OX cells in the 96-well plate. The plate was then incubated at 37°C in 5% CO2. After 72-96 hours, plates were equilibrated to room temperature and cell viability was assessed using the Promega® CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's recommended protocol. Luminescence was measured on a Molecular Devices® FlexStation® 3 multimode microplate reader and analyzed using GraphPad Prism 8.0 software. The half maximal inhibitory concentration (IC50) is the concentration of drug (topotecan or other chemotherapeutic cytotoxic agent) at which the response (cell proliferation) is reduced by 50%.

[0286] Xenograft trials material: Cells: HT1376 (ATCC CRL-1472) human bladder cancer cell line. Mice: 65 5-6 week old female SCID-Biege mice (Charles River). Reagents: G2KT9 anti-ABCG2 x anti-CD4 BsAb produced as described above, human isotype IgG1 (Bioxcell), topotecan.

[0287] method: Cell culture: HT1376 cells were maintained in RPMI medium supplemented with 10% FBS, 1% penicillin, and 1% streptomycin at 37°C and 5% CO. The cell lines used were confirmed to be authentic and mycoplasma negative.

[0288] Seed - 2 x 10 cells diluted in PBS:Matrigel (1:1) 6 Each dose was subcutaneously injected into 50 anesthetized 5-6 week-old female SCID-Biege mice under sterile conditions using a 27G insulin syringe.

[0289] result FIG. 1A shows FACS analysis of the binding of anti-ABCG2 antibodies, G2.65 and G.302, to HEK293 naive cells, HEK293 cells overexpressing human ABCG2 (“hG2”), and HEK293 cells overexpressing cynomolgus monkey ABCG2 (“cG2”).

[0290] The anti-ABCG2 antibody, G2.65, binds to 293 cells overexpressing human ABCG2 ("hG2"), but does not significantly bind to naive 293 cells or 293 cells overexpressing cynomolgus monkey ABCG2 ("cG2").

[0291] The anti-ABCG2 antibody, G2.302, binds to 293 cells overexpressing human ABCG2 (“hG2”) and cynomolgus monkey ABCG2 (“cG2”), but does not significantly bind to naive 293 cells.

[0292] Figure 1B shows that the anti-human ABCG2 antibody 5D3 binds to human ABCG2 expressed in 3T3 and C6 cell lines, as well as to cynomolgus monkey ABCG2 (cG2) expressed by 3T3 cells stably transfected with cABCG2 and to human ABCG2 (hG2) expressed by C6 cells stably transfected with hABCG2.

[0293] 2A-2B. Binding of anti-ABCG2 antibodies to 293 cells overexpressing human ABCG2. Compared to the anti-ABCG2 antibody 5D3, the listed anti-ABCG2 antibodies have lower affinity for ABCG2. Anti-ABCG2 antibodies with lower affinity for ABCG2 compared to 5D3 may be more suitable for (i) creating antibodies that can be conjugated to cytotoxic molecules that preferentially bind to cancer cells relative to non-cancer cells due to the higher expression level of G2 by cancer cells, and / or (ii) creating bispecific antibody molecules that preferentially bind to cells expressing both ABCG2 and a second antigen targeted by the bispecific antibody, while binding significantly less to cells expressing only G2.

[0294] Figure 2C shows the dissociation constants for the indicated anti-G2 antibodies for binding to 293T_ABCG2_OX cells. These antibodies are selected from those listed in Tables 2 and 3. For example, KNJY-G2-420 is another name for the G.420 antibody.

[0295] Figure 2D shows the dissociation constants for the indicated anti-G2 antibodies for binding to 293T_ABCG2_OX cells. These antibodies are selected from those listed in Tables 2 and 3.

[0296] Figure 2E shows the results (EC50 shift) for efflux blockade in 293T.G2 cells by various anti-ABCG2 mAbs (their respective K D enumerated with values).

[0297] Table 3 lists the following characteristics of anti-ABCG2 antibodies: binding to 293T cells stably transfected to express human ABCG2 (293T_ABCG2_OX) measured by FACS, binding affinity to 293T_ABCG2_OX cells (binding = "+", non-significant binding = "-"), binding to cynomolgus monkey ABCG2 (binding = "+", non-significant binding = "-"), blockade of topotecan efflux by 293T_ABCG2_OX cells (efflux blockade = "+", non-significant efflux blocking activity = "-"), effect of the listed antibodies on cell killing by topotecan assessed in 293T_ABCG2_OX cells (killing = "+", non-significant killing = "-"). [Table 4]

[0298] The anti-G2 antibodies G2.640 and G.643, which were generated in rats, did not show significant binding to human or cynomolgus G2 overexpressed in the 293T cell line. The G2.640 antibody has the following sequence: TIFF0007767322000041.tif22165TIFF0007767322000042.tif22164Underlining indicates CDR.

[0299] Table 4 shows the IC50 (nM) of topotecan cytotoxicity in 293T_ABCG2_OX cells in the presence of the listed anti-G2 antibodies. [Table 5]

[0300] FIG. 2F shows the IC50 determination of topotecan cytotoxicity when 293T_ABCG2_OX cells were exposed to anti-G2 antibody.

[0301] Example 2: Bispecific antibodies that bind to ABCG2 and MDR1 This example demonstrates the development of an antibody molecule capable of binding to the extracellular domain of EP. EP blockade results in the resensitization and killing of cells resistant to chemotherapeutic agents. In this example, a bispecific antibody molecule is constructed that binds to the extracellular domains (ECDs) of two EPs, ABCG2 and MDR1. A schematic diagram of the bispecific antibody molecule is shown in Figure 3. Arm A can be the variable heavy chain of an anti-ABCG2 antibody, arm B can be the variable heavy chain of an anti-MDR1 antibody (or anti-TAA antibody), and arm C is a common light chain, which can be derived from an anti-ABCG2 antibody, an anti-MDR1 antibody, a different anti-ABCG2 antibody, a different anti-MDR1 antibody, or an unrelated antibody.

[0302] Materials and Methods Cell lines and cell viability experiments PgP-expressing HEK293T, MCF-7, N6ADR, and SKNF7 cell lines were obtained from the American Type Culture Collection. All cell lines and their derivatives were maintained in RPMI 1640 or DMEM supplemented with up to 10% fetal bovine serum (Sigma), non-essential amino acids, and 2 mmol / L L-glutamine in a humidified incubator at 37°C and 5% CO2 (unless otherwise indicated). Cells were used as supplied, or engineered to overexpress (Ox)PgP, or knocked down (KD) PgP expression with lentivirus-mediated short hairpin RNA, or knocked out (KO) functional genes using CRISPR / Cas-mediated knockout technology essentially as described (Cong, L. et al. (2013) Science 339, 819-823). For IC50 determination of vincristine and paclitaxel, cells were seeded in normal growth medium and allowed to adhere overnight. Paclitaxel, vincristine, or topotecan (Sigma) was added in serial dilutions, and any modulators were added at concentrations ranging from 1 to 500 μM / L. Cell viability was measured after 72 hours using the Celltiter-Glo Luminescent Cell Viability Assay (Promega). The drug concentration resulting in 50% inhibition of cell viability (IC50) was calculated from multiparameter curve analysis (GraphPad Prism software, GraphPad Software, Inc.) and determined from a minimum of two replicates. In the majority of experiments performed, cell lines that did not demonstrate a 50% decrease in cell viability in response to drug and / or modulator treatment were, by definition, deemed to have not reached IC50 and are listed as having an IC50 of >1000 nmol / L for paclitaxel or the drug / modulator combination being tested.

[0303] Recombinant cell lines with stable expression of the described monoclonal antibodies (mAbs) were also generated.

[0304] Cell culture technology and antibody production Standard cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

[0305] 293 and CHO cells were used for transient production of mAb, Fab'2, Fab, and bispecific mAb. Polymer-based cotransfection of Expi293 cells (A14527, ThermoFisher) was used to express the different antibody constructs. Cells were grown in suspension with mammalian expression vectors according to the manufacturer's recommendations.

[0306] For the preparation of bispecific antibody molecules, cells were transfected with the corresponding expression vectors at a ratio of 1:1:4 (heavy chain KK:heavy chain DD:light chain), whereas for standard antibody expression a ratio of 1:2 (heavy chain:light chain) was used.

[0307] Six days after transfection, cells were collected by centrifugation. Specifically, 1 μg of total coding DNA per ml of culture to be transfected was diluted in Opti-MEM® medium (Life Technologies) and incubated with Expifectamine reagent (Life Technologies) in the same medium for 20 minutes. The mixture was then added to Expi293® cells growing in suspension in Expi293® Expression Medium (Life Technologies) at 37°C in air with 8% CO2 at 2.5 million cells / ml. Six days later, the medium containing the antibody construct was collected by centrifugation.

[0308] Reagent cell lines used to test binding, efflux blockade, and cell sensitization to chemotherapeutic agents The 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 μg / mL streptomycin at 37°C with 5% CO incubation.

[0309] 293T cells were transiently transfected with a human P-glycoprotein-tagged ORF clone in the pLenti-C-Myc-DDK-P2A-Puro plasmid using an optimized PEIPro™ transfection protocol (Polyplus). DNA and JetPEI® were diluted in culture medium and then gently mixed for approximately 10 minutes. This mixing resulted in the formation of a transfection complex, which was added directly to the cell culture. Efflux blockade was measured using a multidrug-resistant direct dye efflux assay (Chemicon) according to the manufacturer's protocol.

[0310] Sources of target sequences, antibody sequences, and specific anti-target antibody sequences P-glycoprotein (PgP), also known as multidrug resistance protein 1 (MDR1), (gene ABCB1) (NM_000927) tagged ORF clone in pLenti-C-Myc-DDK-P2A-Puro, and ABCG2 ABCG2 (NM_004827) human tagged ORF clone in pCMV6-XL5 were obtained from Origene, anti-CD47 antibody (CC2C6, Seiffert M, et al. (1999) Blood 94:3633) from Biolegend, anti-ABCB1 JSB-1 (MAB4120) from Millipore, and anti-human ABCG2 antibody (clone 5D3) from R&D Systems. MDR1 is also referred to herein as KPB1. ABCG2 is also referred to herein as KPG2.

[0311] Generation of stable ABCB1-overexpressing (Ox) cell lines To characterize both binding and in vitro efficacy, we developed a cell line stably overexpressing ABCB1. We utilized adherent 293T naive cells obtained from the American Type Culture Collection (ATCC). This cell line endogenously expresses ABCB1 at low to moderate levels on the cell surface, as characterized by flow cytometry using a commercially available ABCB1 antibody (Biolegend, clone 4E3.16). 293T naive cells were transfected with ABCB1 using Polyplus PEIpro reagent. Three days after transfection, cells were placed under selection using hygromycin B solution (Millipore, Sigma). After 14 days of continuous hygromycin B selection, 293T cells were assessed for ABCB1 cell surface expression. To ensure that untransfected cells were not expanded in subsequent cultures, bulk sorting of ABCB1-positive 293T cells using fluorescence-activated cell sorting (FACS) was performed using a FACSAriaI (BD Biosciences). Bulk-sorted 293T ABCB1-overexpressing cells were expanded and ABCB1 overexpression was subsequently reconfirmed.

[0312] Generation of stable ABCG2-overexpressing (Ox) cell lines To characterize both binding and in vitro efficacy, we developed cell lines stably overexpressing ABCG2. We utilized adherent 293T, 3T3, and C6 naive cells obtained from the American Type Culture Collection (ATCC). These cell lines endogenously express low to moderate levels of ABCG2 on the cell surface, as characterized by flow cytometry using a commercially available ABCG2 antibody (R&D Systems, clone 5D3). 293T naive cells were transfected with ABCG2 using Lipofectamine 3000 reagent, while 3T3 and C6 were transfected using neon electroporation. Three days after transfection, cells were placed under selection using hygromycin B solution (Millipore, Sigma). After 14 days of continuous hygromycin B selection, transfected cells were assessed for ABCG2 cell surface expression. Stable ABCG2 cells were single-sorted using a FACSAriaI (BD Biosciences) and expanded, and ABCG2 overexpression was subsequently reconfirmed.

[0313] Generation of stable ABCB1 KD 293T cell lines To characterize both binding and in vitro efficacy, we developed a cell line with stable knockdown of ABCB1 expression. Lentivirus was generated in naive 293T cells by transfection with the GE Dharmacon GIPZ lentiviral vector containing the R8.74 helper plasmid, VSVG envelope plasmid, and shRNA against ABCB1. The collected lentivirus was then used to transduce adherent naive 293T cells. Three days after transduction, the transduced 293T cells were assessed for ABCB1 cell surface expression by flow cytometry (Biolegend, clone 4E3.16). Compared to naive 293T cells, which endogenously express ABCB1 at low levels, the transduced 293T cells had no ABCB1 expression. In addition, the GIPZ lentiviral vector contains GFP. All transduced 293T cells were GFP+, indicating successful transduction, along with the reduced expression. The lack of ABCB1 expression was reconfirmed by flow cytometry in subsequent passages.

[0314] Generation of KO cell lines To construct the ABCG2 gene knockout HEK293 cell line, HEK293 host cells were first cultured in adherent culture in DMEM (Dulbecco's Modified Eagle's 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.

[0315] gRNA design was performed using the online CHOPCHOP web tool to select target sites for CRISPR / Cas9, CRISPR / Cpf1, or TALEN-directed mutagenesis (see 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).

[0316] 293T cells were transfected by lipid-based transfection using CRISPRMax reagent (ThermoFisher) according to the manufacturer's protocol. Briefly, 1 day before transfection, adherent cells were transfected at 0.2 × 10 cells per well. 5 Cells were seeded into 96-well plates at 1000 x g / ml. On the day of transfection, a solution of GeneArt Platinum Cas9 protein, gRNA, and transfection reagent was added to the cells. Seventy-two hours after transfection, single-cell selection by limiting dilution was followed by cell culture in a 96-well plate format for two weeks. Subsequently, picked clones were passaged into 24-well plates and tested by genotype confirmation 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 had resulted in indels in one allele (monoallelic) or both alleles (biallelic) in singly isolated clones. Expression of the protein of interest in clones with mutations in both alleles was tested by FACS.

[0317] Construction of the sequences of the molecules tested (human Fc, mouse Fv, or human Fc, or humanized Fv) Expression vectors: For the generation of antibody expression vectors, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with either the human IgG1 constant heavy chain or the human IgG1 kappa constant light chain, respectively, previously inserted into a common recipient expression vector optimized for expression in mammalian cell lines. The gene to be expressed was cloned into the pCI-neo mammalian expression vector (Promega), which uses the full-length human cytomegalovirus (CMV) immediate-early promoter for high-level gene expression. The two antibody chains were cloned into two different vectors.

[0318] The N-terminal signal sequences from mouse IgG heavy and kappa light chains were used for secretory expression of the heavy and light chains, respectively. The signal peptides were cleaved during expression, leaving intact N-termini. In the Fab constructs, the C-terminus of the CH1 IgG1 constant region was fused to a 6xHis tag to facilitate purification.

[0319] For the generation of bispecific antibody vectors, IgG1-derived bispecific molecules contain at least two antigen-binding moieties capable of specifically binding to two different targets, namely PgP (ABCB1) and ABCG2. The antigen-binding moieties are Fab fragments consisting of heavy and light chains, each containing a variable and constant region. A common light chain was identified that could pair to provide acceptable binding as both the Fabs of Fab anti-PgP (aPgP) and Fab anti-ABCG2 (aABCG2), and its use allowed for avoiding mispairing of the LC. The bispecific constructs were generated based on the electrostatic steering effect (see, e.g., Gunasekeran et al., (2010) Journal of Biological Chemistry 285, 19637-19646, the entire disclosure of which is incorporated herein by reference). Briefly, polypeptide chains or half antibodies against the target were assembled as bispecific antibodies through charge-pair substitutions in the CH3 domain, i.e., one heavy chain contained K392D and K409D substitutions ("DD") and the other contained E356K and D399K substitutions ("KK").

[0320] The bispecific mAb molecule contained a variable heavy chain containing HCDRs 1-3 from the anti-MDR1 antibody 15D3, a variable heavy chain containing HCDRs 1-3 from the anti-G2 antibody G2-255, and a common light chain kappa sequence derived from the anti-MDR1 antibody MRK16, both on a human IgG1 Fc.

[0321] The monoclonal antibodies 15D3 (see, e.g., U.S. Pat. No. 5,959,084, the disclosure of which is incorporated herein by reference in its entirety) and MRK16 (Iwahashi et al., Cancer Research 53, 1993, the disclosure of which is incorporated herein by reference in its entirety) previously generated against PgP were cloned into human IgG1 / kappa expression vectors as recombinant engineered antibodies.

[0322] Variable heavy and light chain fragments from mouse hybridoma sequences were available and cloned in the same background of leader sequences and constant regions.

[0323] The anti-ABCG2 monoclonal antibody sequences are disclosed in Table 2, and the antibody variable heavy and light chain fragments were cloned in the same background of leader sequences and constant regions in two separate vectors.

[0324] result Detection of ABCB1 and ABCG2 specific binding The binding specificity of the mAb, Fab, and bispecific IgG1 was tested by FACS using naive 293T cells overexpressing the human ABCB1 target and 293T cells overexpressing human ABCG2. Briefly, different cell lines were incubated with various amounts of mAb or bispecific mAb, or a human IgG1 isotype control antibody, on ice for 1 hour. Cells were washed three times with FACS buffer (PBS containing 0.5% BSA). Alexa647-labeled goat anti-human antibody was added as a secondary antibody, and the samples were incubated for an additional hour on ice. The samples were washed and analyzed using a BD FACS Canto (BD Biosciences).

[0325] The binding specificities of the mAb, Fab, and bispecific IgG1 were tested by FACS using the 293T cell line, 293T naive cells overexpressing the human ABCB1 target, and 293T cells overexpressing human ABCG2. Briefly, different cell lines were incubated with various amounts of mAb or bispecific mAb, or a human IgG1 isotype control antibody, on ice for 1 hour. Cells were washed three times with FACS buffer (PBS containing 0.5% BSA). Alexa647-labeled goat anti-human antibody was added as a secondary antibody, and the samples were incubated for an additional hour on ice. The samples were washed and analyzed using a BD FACS Canto (BD Biosciences).

[0326] This example demonstrates the construction of a bispecific heterobivalent antibody molecule in which one arm binds to the efflux pump MDR1 / PgP and the other arm binds to the efflux pump ABCG2. When both targets are simultaneously present on the surface of a cell, the bispecific antibody binds to the cell with relatively high affinity / avidity. In comparison, when either MDR1 / PgP or ABCG2 is absent or substantially reduced, bispecific antibody binding is significantly reduced or undetectable.

[0327] The bispecific antibody contains one arm that binds to and antagonizes a transporter protein (the efflux pump PgP), making cells more sensitive to chemotherapeutic agents, while the other arm binds to another efflux pump, ABCG2.

[0328] Example 3: Bispecific antibodies that bind to ABCG2 and EGFR This example demonstrates the development of antibody molecules capable of binding to the extracellular domains of ABCG2 and EGFR. EP blockade results in the resensitization and killing of cells resistant to chemotherapeutic agents.

[0329] Materials and methods were similar to those described in Example 2. 293T cells were transiently transfected with the ABCG2 (NM_004827) human tagged ORF clone in the pCMV6-XL5 plasmid using an optimized PEIPro™ transfection protocol (Polyplus). For the generation of bispecific antibody vectors, IgG1-derived bispecific antibody molecules contain at least two antigen-binding moieties capable of specifically binding to two different targets: ABCG2 and EGFR. The antigen-binding moieties are Fab fragments consisting of heavy and light chains, each containing a variable and constant region. A common light chain was identified that could pair to provide acceptable binding as both Fab anti-EGFR and Fab anti-ABCG2 (aABCG2).

[0330] Figure 5. FACS analysis showing that both ABCG2 and EGFR are expressed in A549 cells. Cells were stained with PE-conjugated anti-human ABCG2 antibody, clone 5D3, and PE-conjugated anti-human EGFR antibody, clone AY13.

[0331] The anti-ABCG2 KNJY-G2-65 antibody variable heavy and light chain fragments were cloned in the same background of leader sequences and constant regions in two separate vectors.

[0332] The sequence and corresponding structure of the anti-EGFR antibody 6B3S (cetuximab) are available at https: / / www.ncbi.nlm.nih.gov / Structure / pdb / 6B3S (Molecular Basis for Necitumumab Inhibition of EGFR Variants Associated with Acquired Cetuximab Resistance, Bagchi A, Haidar JN, Eastman SW, Vieth M, Topper M, Iacolina MD, Walker JM, Forest A, Shen Y, Novosiadly RD, Ferguson KM, Mol. Cancer Ther. (2018) 17 p. 521-531), and the sequence is reproduced herein for reference. The 6B3S antibody variable heavy chain sequence is as follows: The TIFF0007767322000044.tif211656B3S antibody variable light chain sequence is as follows: TIFF0007767322000045.tif17165The CDRs in the antibody according to Kabat nomenclature are shown in bold and underlined.

[0333] result Detection of EGFR and ABCG2 specific binding The binding specificity of EGFR-targeting mAbs, Fabs, and bispecific IgG1s was tested by ELISA using rhEGFR-his tagged protein (Sino Biological). Briefly, microtiter plates were coated with 50 μl of purified human EGFR-his protein at 2 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 serial dilutions and incubated for 1 hour at room temperature. Anti-EGFR antibody 6B3S was used as a positive control, and human IgG1 was used as an isotype control. Subsequently, the plates were washed three times with PBS / Tween and then incubated with HRP-conjugated donkey anti-human constant region-specific secondary reagent for 1 hour at room temperature. After washing, the plates were developed with HRP substrate. The reaction was stopped with 2 M H2SO4, and the OD was measured at 520 nM.

[0334] The binding specificity of the mAb, Fab, and bispecific IgG1 was tested by FACS using naive 293T cells overexpressing human ABCG2 and naive A549 cells naturally expressing EGFR and ABCG2. Briefly, different cell lines were incubated with various amounts of mAb or bispecific mAb, or a human IgG1 isotype control antibody, on ice for 1 hour. Cells were washed three times with FACS buffer (PBS containing 0.5% BSA). Alexa647-labeled goat anti-human antibody was added as a secondary antibody, and the samples were incubated for an additional hour on ice. The samples were washed and analyzed using a BD FACS Canto (BD Biosciences).

[0335] Figure 6 provides binding data for two different bispecific mAbs that bind to EGFR and ABCG2. aEGFR refers to the anti-EGFR antibody 6B3S. The bispecific antibody: aEGFR DD HC / G2.173KK HC / G.173 LC contains the variable heavy chain from the anti-EGFR antibody 6B3S fused to human IgG1 with a DD substitution, the variable heavy chain from the anti-ABCG2 antibody G2.173 fused to human IgG1 with a KK substitution, and two copies of the variable light chain of the anti-ABCG2 antibody G2.173 fused to a kappa sequence. The bispecific antibody: aEGFR DD HC / G2.65KK HC / G.65 LC contains the variable heavy chain from 6B3S fused to human IgG1 with a DD substitution, the variable heavy chain from the anti-ABCG2 antibody G2.65 fused to human IgG1 with a KK substitution, and two copies of the variable light chain of the anti-ABCG2 antibody G2.65 fused to a kappa sequence.

[0336] This example demonstrates the construction of a bispecific heterobivalent antibody molecule in which one arm binds to the efflux pump ABCG2 and the other arm binds to an EGFR target. When both targets are simultaneously present on the cell surface, the bispecific antibody binds to the cell with relatively high affinity / avidity. As shown in Figure 6, the G2.6.5-based bispecific antibody has several advantages in the binding studies shown: it demonstrates stronger affinity for EGFR (by ELISA) and stronger binding to A549 cells displaying both targets; when the ABCG2 efflux pump is overexpressed, both bispecific antibodies bind similarly.

[0337] Figure 7 shows the binding of the indicated anti-ABCG2 monoclonal antibodies to 293T cells overexpressing human ABCG2 ("hG2") and cynomolgus ABCG2 (cG2), respectively. As indicated by the dissociation constants (Kd), antibodies G2.748, G2.757, G2.758, and G2.760 bind to both human and cynomolgus ABCG2 with excellent binding affinity.

[0338] Figure 8 shows the binding of anti-ABCG2 antibody G2.748 to 293T cells overexpressing human or cynomolgus ABCG2, along with the corresponding dissociation constants (Kd) compared to anti-ABCG2 antibody 5D3. As the Kd values ​​indicate, G2.748 binds to both human and cynomolgus ABCG2 with excellent binding affinity.

[0339] Figures 9-11 show the binding of various recombinant anti-ABCG2 antibodies (Panels 1-3) to 293T cells overexpressing human and cynomolgus ABCG2, respectively, and the corresponding Kd values. Of particular note is the strong binding of antibodies G2.636, G2.631, and G2.643 to both human and cynomolgus ABCG2.

[0340] 12 and 13 show the results of testing the listed recombinant anti-ABCG2 antibodies for efflux inhibitory activity, using the small molecule ABCG2 inhibitors fumitremordin C (FTC) and Ko143 as positive controls, according to the previously described efflux blockade experimental procedure. In particular, antibodies G2.636, G2.643, G2.333, G2.631, and G2.318 exhibited potent efflux inhibitory activity, with G2.636 and G2.643 being particularly effective.

[0341] Figure 14 shows the effect of anti-ABCG2 antibodies G2.643, G2.420, and G2.631 on topotecan cytotoxicity in 293T_ABCG2_OX cells, 293T cells stably transfected to express ABCG2, using FTC and 5D3 as positive controls. All three antibodies tested significantly increased the cytotoxic activity of topotecan.

[0342] Figure 15 shows that the combination of anti-ABCG2 antibodies G2.343, G2.636, and G2.629 with topotecan was effective in reducing tumor volume in a xenograft study using topotecan-resistant Panc1 / T300 cells. PANC1 (ATCC, CRL-1469) is a pancreatic ductal carcinoma cell line. Topotecan-resistant Panc1 / T300 cells were developed by sequentially incubating PANC1 cells with culture medium containing increasing concentrations of topotecan: 25, 50, 100, 200, and 300 nM. Selected cells showed elevated ABCG2 expression and were resistant to topotecan compared to parental PANC1 cells. Arrows indicate the dosing schedule.

[0343] Figure 16 shows that the combination of anti-ABCG2 antibodies G2.343 and G2.631 with topotecan is effective in reducing tumor volume in a xenograft study using the non-small cell lung cancer (NSCLC) epithelial cancer cell line A549 (ATCC, CCL-185). Arrows indicate the dosing schedule. While all combinations reduce tumor volume, the G2.343 / topotecan combination is shown to be particularly effective.

[0344] Figure 17 shows the efficacy of the anti-ABCG2 antibody G2.333 administered alone or in combination with camptothecin-11 (CPT11, irinotecan) in a xenograft study using the non-small cell lung cancer (NSCLC) epithelial cancer cell line A549 (ATCC, CCL-185). Arrows indicate the dosing schedule. The efficacy of the G2.33 / CPT11 combination significantly exceeds that of the 5D3 / CPT11 combination in this experiment.

[0345] Figure 18 shows the efficacy of the bispecific anti-ABCG2 antibody G2.318 / KT3 / G2.318, administered alone or in combination with topotecan, in a xenograft study using topotecan-resistant Panc1 / T300 cells. Arrows indicate the dosing schedule. KT3 = cetuximab, an anti-EGFR antibody. The tested bispecific antibody significantly reduced tumor volume both as a single agent and in combination with topotecan.

[0346] Figure 19 shows the efficacy of the bispecific anti-ABCG2 antibody G2.318 / KT9 / G2.318 administered alone or in combination with topotecan in a xenograft study using the HT1376 (ATCC, CRL-1472) bladder epithelial cancer cell line. Arrows indicate the dosing schedule. KT9 = atezolizumab, an anti-PD-L1 antibody. The combination of the tested bispecific antibody with topotecan has been shown to be effective in reducing tumor volume.

[0347] Figure 20 shows the efflux inhibitory activity and binding of various humanized G2.636 anti-ABCG2 antibodies to human and cynomolgus monkey ABCG2. All humanized variants of the anti-ABCG2 antibody G2.636 retain activity, with the G2.636.hu47 variant showing particularly superior expression and activity.

[0348] Figure 21 shows the schematic structures of two humanized ABCG2 / CD47 bispecific antibodies (5F9huscFv-G2.318.hu33 and B6H12huscFv-G2.318.hu33) and their binding to human and cynomolgus monkey ABCG2 compared to G2.318.hu33 and 5D3, respectively. Both humanized antibodies show excellent binding.

[0349] Figure 22 shows the schematic structure of the humanized ABCG2 / HER2 bispecific antibody KT1scFv-G2.318.hu33 and its binding to human ABCG2 and human HER2, respectively. KT1 = HER2. The bispecific antibody shows strong binding to both ABCG2 and HER2.

[0350] Figures 23A-23C show the schematic structure of the bispecific antibody G2.318KK KT9DD G2.318 and its binding to ABCG2+KT9- (Figure 23A), ABCG2-KT9+ (Figure 23B), and ABCG2+KT9+293T (Figure 23C) cells. KT9 = atezolizumab, an anti-PD-L-1 monoclonal antibody. The antibody shows excellent binding to both ABCG2 and PD-L1.

[0351] Figures 24A and 24B show the binding of the G2.643 antibody and its humanized version, G2.643.hu46, to human and cynomolgus ABCG2 (Figure 24A), and the efflux inhibitory activity of the G2.643 and G2.643.hu46 antibodies (Figure 24B).

[0352] The present disclosure includes the following embodiments. Embodiment 1 An antibody that specifically binds to ATP-binding cassette subfamily G member 2 (ABCG2), wherein the antibody has the following binding characteristics with respect to ABCG2: An antibody that competes with an antibody comprising heavy chain complementarity-determining regions 1 to 3 (HCDR1 to 3) and light chain CDR1 to 3 (LCDR1 to 3) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. Embodiment 2 The antibody of embodiment 1, comprising HCDRs 1 to 3 of the VH region of an antibody listed in Table 2. Embodiment 3 The antibody of embodiment 2, comprising LCDRs 1 to 3 of the VL region of an antibody listed in Table 2. Embodiment 4 The antibody of embodiment 1, comprising the heavy chain complementarity determining regions (HCDRs) and light chain CDRs (LCDRs) of the paired variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. Embodiment 5 An antibody molecule that specifically binds to ATP-binding cassette subfamily G member 2 (ABCG2), wherein the antibody comprises heavy chain complementarity-determining regions 1-3 (HCDR1-3) and / or light chain CDR1-3 (LCDR1-3) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. Embodiment 6 The antibody molecule of embodiment 5, wherein the antibody comprises HCDRs 1-3 and LCDRs 1-3 of pairs of VH and VL domains of an antibody listed in Table 2. Embodiment 7 The antibody of embodiment 5, wherein the antibody comprises HCDRs 1-3 of the VH region of a first antibody listed in Table 2. Embodiment 8 The antibody molecule of embodiment 7, wherein the antibody comprises LCDRs 1 to 3 of the VL region of a second antibody in Table 2. Embodiment 9 The antibody molecule of embodiment 5, wherein the antibody molecule comprises the variable light (VL) chain and / or variable heavy (VH) chain of an antibody listed in Table 2. Embodiment 10 2. The antibody molecule of any one of the preceding embodiments, wherein the antibody inhibits ABCG2-mediated efflux when bound to a cell expressing ABCG2. Embodiment 11 10. The antibody molecule of any one of the preceding embodiments, wherein the antibody comprises a humanized light chain. Embodiment 12 The antibody molecule of any one of the preceding embodiments, wherein the antibody comprises a humanized heavy chain. Embodiment 13 The antibody may be a bispecific antibody, an Ig monomer, a Fab fragment, a F(ab') 2 10. The antibody molecule of any one of the preceding embodiments, wherein the antibody molecule is selected from the group consisting of: a Fd fragment, an scFv, an scAb, a dAb, and an Fv. Embodiment 14 13. The antibody molecule of any one of embodiments 1 to 12, wherein the antibody comprises a VL region and a VH region that are present in separate polypeptides. Embodiment 15 13. The antibody molecule of any one of embodiments 1 to 12, wherein the antibody comprises a VL region and a VH region present in a single polypeptide. Embodiment 16 A bispecific antibody molecule that binds to ATP-binding cassette subfamily G member 2 (ABCG2) and multidrug resistance protein 1 (MDR1), wherein the antibody molecule comprises two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain, wherein the VL chains each comprise an antigen-binding site for MDR1, the first VH chain comprises an antigen-binding site for MDR1, and the second VH chain comprises an antigen-binding site for ABCG2, and the second VH chain binds to ABCG2 when paired with one of the light chains. Embodiment 17 the first VH chain has the amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 1 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVS (SEQ ID NO: 418) (wherein X 1 is N, Q, or S), or 17. The bispecific antibody molecule of embodiment 16, wherein the first VH chain comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 418. Embodiment 18 17. The bispecific antibody molecule of embodiment 16, wherein the first VH chain comprises heavy chain complementarity determining regions 1 to 3 (HCDR1-3) of a VH chain from an anti-MDR1 antibody, wherein HCDR1 comprises the sequence: GFTFSRYTMS (SEQ ID NO: 419), HCDR2 comprises the sequence: VATISSGGGNTYYPDSVKG (SEQ ID NO: 362), VATISSGGGQTYYPDSVKG (SEQ ID NO: 363), or VATISSGGGSTYYPDSVKG (SEQ ID NO: 364), and HCDR3 comprises the sequence: ARYGAGDAWFAY (SEQ ID NO: 365). Embodiment 19 19. The bispecific antibody molecule of any one of embodiments 16 to 18, wherein the second VH chain comprises heavy chain complementarity-determining regions 1 to 3 (HCDRs 1 to 3) of the VH chain of an anti-ABCG2 antibody having a sequence as shown in Table 2. Embodiment 20 the second VH chain comprises HCDRs 1 to 3 of the VH chain of an anti-ABCG2 antibody, wherein the HCDR1 comprises the sequence: NNAMS (SEQ ID NO: 82), the HCDR2 comprises the sequence: TITGGGSYTYYPDSVKG (SEQ ID NO: 112), and the HCDR3 comprises the sequence: PDGNYEGVLAY (SEQ ID NO: 154); or the second VH chain has the amino acid sequence: 20. The bispecific antibody molecule of any one of embodiments 16 to 19, comprising an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% identity to EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQTPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVSA (SEQ ID NO: 13). Embodiment 21 21. The bispecific antibody molecule of any one of embodiments 16 to 20, wherein the two identical VL chains comprise the light chain CDR1-3 (LCDR1-3) of the VL chain of an anti-MDR1 antibody having the amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367). Embodiment 22 the two identical VL chains comprise light chain CDRs 1 to 3 (LCDRs 1 to 3) of an anti-MDR1 antibody; (i) the LCDR1 comprises the sequence: RSSQSIVHSTGNTYLE (SEQ ID NO: 368); (ii) the LCDR2 comprises the sequence: KVSNRFS (SEQ ID NO: 305); (iii) the LCDR3 comprises the sequence: QGSHFPRT (SEQ ID NO: 369); or the VL chain has the amino acid sequence: 22. The bispecific antibody molecule of any one of embodiments 16 to 21, comprising an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% identity to DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367). Embodiment 23 The two identical VL chains have the amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 2 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) (wherein X 2 is N, Q, or S), or the VL chain has the amino acid sequence: DVLMTQTPVSLSSVSLGDQASISCRSSQSIVHSTGX 2 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) (wherein X2 is N, Q, or S). Embodiment 24 the two identical VL chains comprise light chain CDRs 1 to 3 (LCDRs 1 to 3) of an anti-MDR1 antibody; (i) the LCDR1 has the sequence: RSSQSIVHSTGX 2 TYLE (SEQ ID NO: 371), (ii) the LCDR2 comprises the sequence: KISNRFS (SEQ ID NO: 372); (iii) the LCDR3 comprises the sequence: FQASHFPRT (SEQ ID NO: 373); where X 2 is N, Q, or S. Embodiment 25 A bispecific antibody molecule that binds to ATP-binding cassette subfamily G member 2 (ABCG2) and multidrug resistance protein 1 (MDR1), the antibody molecule comprising two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain, each of the VL chains comprising an antigen-binding site for ABCG2, the first VH chain comprising an antigen-binding site for MDR1, and the second VH chain comprising an antigen-binding site for ABCG2, and the first VH chain binds to MDR1 when paired with one of the light chains. Embodiment 26 the first VH chain has the amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 1 TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVS (SEQ ID NO: 418) (wherein X 1 is N, Q, or S). Embodiment 27 26. The bispecific antibody molecule of embodiment 25, wherein the first VH chain comprises heavy chain complementarity determining regions 1 to 3 (HCDR1-3) of a VH chain from an anti-MDR1 antibody, wherein HCDR1 comprises the sequence: GFTFSRYTMS (SEQ ID NO: 419), HCDR2 comprises the sequence: VATISSGGGNTYYPDSVKG (SEQ ID NO: 362), VATISSGGGQTYYPDSVKG (SEQ ID NO: 363), or VATISSGGGSTYYPDSVKG (SEQ ID NO: 364), and HCDR3 comprises the sequence: ARYGAGDAWFAY (SEQ ID NO: 365). Embodiment 28 28. The bispecific antibody molecule of any one of embodiments 25 to 27, wherein the second VH chain comprises heavy chain complementarity-determining regions 1 to 3 (HCDRs 1 to 3) of the VH chain of an anti-ABCG2 antibody having a sequence as shown in Table 2. Embodiment 29 29. The bispecific antibody molecule of any one of embodiments 25 to 28, wherein the two identical VL chains comprise the light chain CDRs 1 to 3 (LCDRs 1 to 3) of the VL chains of an anti-ABCG2 antibody listed in Table 2. Embodiment 30 30. The bispecific antibody of any one of embodiments 16 to 29, wherein said antibody inhibits the activity of at least one of ABCG2 and MDR1. Embodiment 31 A bispecific antibody molecule that binds to ATP-binding cassette subfamily G member 2 (ABCG2) and a tumor-associated antigen (TAA), the antibody molecule comprising two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain; the VL chains each comprise an antigen-binding site for ABCG2, the first VH chain comprises an antigen-binding site for ABCG2, and the second VH chain comprises an antigen-binding site for the TAA, and the second VH chain binds to the TAA when paired with one of the light chains; or a bispecific antibody molecule, wherein the VL chains each comprise an antigen-binding site for the TAA, the first VH chain comprises an antigen-binding site for ABCG2, and the second VH chain comprises an antigen-binding site for the TAA, and the first VH chain binds to ABCG2 when paired with one of the light chains. Embodiment 32 32. The bispecific antibody molecule of embodiment 31, wherein each of the VL chains comprises an antigen-binding site for ABCG2, the first VH chain comprises an antigen-binding site for ABCG2, and the second VH chain comprises an antigen-binding site for the TAA, and the second VH chain binds to the TAA when paired with one of the light chains. Embodiment 33 33. The bispecific antibody molecule of embodiment 32, wherein the first VH chain comprises heavy chain complementarity determining regions 1-3 (HCDRs 1-3) of an anti-ABCG2 antibody listed in Table 2. Embodiment 34 the first VH chain comprises heavy chain complementarity determining regions 1 to 3 (HCDR1-3), wherein HCDR1 comprises the sequence: DDYVH, HCDR2 comprises the sequence: RIDPANGNTRYAPKFRG (SEQ ID NO: 115), and HCDR3 comprises the sequence: PLWVGGFAY (SEQ ID NO: 157); or the first VH chain comprises the amino acid sequence: QVQLQQSGADLVRPGASVKLSCTASGFNIKDDYVHWVKQRPEQGLEWIGRIDPANGNTRYAPKFRGKATMTADTSSNTAYLQLSSLTSADTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 16), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPANGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 17), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPAQGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 18), or 34. The bispecific antibody molecule of embodiment 32 or 33, comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPASGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 19). Embodiment 35 35. The bispecific antibody molecule of any one of embodiments 32 to 34, wherein the antigen-binding sites of the two VL chains comprise the light chain CDRs 1 to 3 (LCDRs 1 to 3) of an antibody listed in Table 2. Embodiment 36 the antigen-binding sites of the two VL chains comprise an LCDR1 comprising the sequence: RSSQSLVHSDVNTYLH (SEQ ID NO: 270), an LCDR2 comprising the sequence: KVSNRFS (SEQ ID NO: 305), and an LCDR3 comprising the sequence: SQTTHVPYT (SEQ ID NO: 334); or the VL chain comprises the amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDVNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVESEDLGIYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 199), or 36. The bispecific antibody molecule of any one of embodiments 32 to 35, comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDVNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 200). Embodiment 37 the first VH chain comprises heavy chain complementarity determining regions 1 to 3 (HCDR1 to 3), wherein the HCDR1 comprises the sequence: SGYIS (SEQ ID NO: 84), the HCDR2 comprises the sequence: WIYAGTGISNFNQKFTG (SEQ ID NO: 114), and the HCDR3 comprises the sequence: GARKTLDF (SEQ ID NO: 156); or the first VH chain comprises the amino acid sequence: 34. The bispecific antibody molecule of embodiment 32 or 33, comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to QGQMHQSGAELVKPGASVKLSCKTSGFTFNSGYISWLKQKPRQSLEWIAWIYAGTGISNFNQKFTGKAQLTVDTSSSTAYMQLSSLTSADSAIYFCASGARKTLDFWGQGTSVTVSS (SEQ ID NO: 15). Embodiment 38 38. The bispecific antibody molecule of embodiment 37, wherein the antigen-binding sites of the two VL chains comprise the light chain CDRs 1-3 (LCDRs 1-3) of an antibody listed in Table 2. Embodiment 39 the antigen-binding sites of the two VL chains comprise an LCDR1 comprising the sequence: KASDQINYWLA (SEQ ID NO: 269), an LCDR2 comprising the sequence: GATSLET (SEQ ID NO: 10), and an LCDR3 comprising the sequence: QQYWTTPYT (SEQ ID NO: 333); or the VL chain comprises the amino acid sequence: 39. The bispecific antibody molecule of embodiment 37 or 38, comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to DIQMTQSSSYLSVSVGGRVTITCKASDQINYWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSFQTEDVATYYCQQYWTTPYTFGGGTKVEIK (SEQ ID NO: 198). Embodiment 40 40. The bispecific antibody molecule of any one of embodiments 32 to 39, wherein the TAA is EGFR and the second VH chain comprises heavy chain complementarity determining regions 1 to 3 (HCDRs 1 to 3) of the VH chain of the 6B3S antibody comprising the amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 388). Embodiment 41 33. The bispecific antibody molecule of embodiment 32, comprising a combination of a first VH chain comprising HCDRs 1-3, a second VH chain comprising HCDRs 1-3, and a common VL chain comprising LCDRs 1-3 as shown in the table below. TIFF0007767322000046.tif90156 Embodiment 42 42. The bispecific antibody molecule of any one of embodiments 16 to 41, wherein the antibody comprises a humanized light chain. Embodiment 43 42. The bispecific antibody molecule of any one of embodiments 16 to 41, wherein the antibody comprises a humanized heavy chain. Embodiment 44 44. The antibody molecule of any one of embodiments 1 to 15, or the bispecific antibody molecule of any one of embodiments 16 to 43, for use in a method for treating cancer in a subject, wherein the method comprises administering the antibody to said subject. Embodiment 45 45. The antibody molecule or bispecific antibody molecule for use according to embodiment 44, wherein the method comprises administering the antibody in combination with at least one additional active agent, wherein the at least one additional active agent comprises a chemotherapeutic agent, an inhibitor of a multidrug resistance transporter, an immunotherapeutic agent, or a combination thereof. Embodiment 46 46. ​​The antibody molecule or bispecific antibody molecule for use according to embodiment 45, wherein said at least one additional active agent is a chemotherapeutic agent, optionally wherein said chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate. Embodiment 47 47. The antibody molecule or bispecific antibody molecule for use according to any one of embodiments 44 to 46, wherein the subject to be treated has a cancer that has been determined to be resistant to treatment with said chemotherapeutic agent. Embodiment 48 1. A pharmaceutical composition comprising: an antibody of any one of the preceding embodiments; and and a pharmaceutically acceptable excipient. Embodiment 49 49. The pharmaceutical composition of embodiment 48, further comprising an additional active agent. Embodiment 50 The pharmaceutical composition of embodiment 48, wherein the additional active agent is a chemotherapeutic agent. Embodiment 51 The pharmaceutical composition of embodiment 50, wherein the additional active agent comprises an inhibitor of a multidrug resistance transporter. Embodiment 52 The pharmaceutical composition of embodiment 50, wherein the additional active agent comprises an immunotherapeutic agent. Embodiment 53 One or more nucleic acids comprising one or more sequences encoding the antibody molecule of any one of embodiments 1 to 15 or the bispecific antibody molecule of any one of embodiments 16 to 43. EMBODIMENT 54 One or more recombinant expression vectors comprising one or more nucleic acids of embodiment 53. Embodiment 55 A host cell genetically modified with one or more recombinant expression vectors of embodiment 54. Embodiment 56 An immune effector cell comprising a chimeric antigen receptor (CAR) comprising an ABCG2-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the ABCG2-binding domain comprises heavy chain complementarity-determining regions 1-3 (HCDR1-3) and / or light chain CDR1-3 (LCDR1-3) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of an antibody listed in Table 2. Embodiment 57 16. A method for assaying expression of ABCG2 on the cell surface of a cell, comprising contacting said cell with the antibody of any one of embodiments 1 to 15. Embodiment 58 58. The method of embodiment 57, wherein the antibody is detectably labeled. Embodiment 59 16. A method for inhibiting the efflux activity of ABCG2 expressed by a living cell, the method comprising contacting said cell with the antibody of any one of embodiments 1 to 15. Embodiment 60 59. The method of embodiment 58, further comprising contacting the cells with an inhibitor of MDR1-mediated efflux. Embodiment 61 61. The method of embodiment 59 or 60, further comprising contacting said cells with a chemotherapeutic agent. Embodiment 62 62. The method of any one of embodiments 59 to 61, wherein the cells are cancer cells. Embodiment 63 63. The method of embodiment 62, wherein the cancer cells are multidrug resistant cancer cells. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the invention that certain changes and modifications can be made therein without departing from the spirit or scope of the appended claims.

[0353] Thus, the foregoing merely illustrates the principles of the present invention. It should be understood that those skilled in the art can devise various configurations, not explicitly described or shown herein, that embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the present technology, and should be construed as not being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly recited in the claims.

[0354] Accordingly, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being incorporated for a claim limitation only if the precise phrase "means for" or the precise phrase "step for" appears at the beginning of such claim limitation; if such precise phrases are not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is not incorporated.

Claims

1. A bispecific antibody molecule that binds to ATP-binding cassette subfamily G member 2 (ABCG2) and (i) a tumor-associated antigen (TAA) or (ii) multidrug resistance protein 1 (MDR1), wherein the bispecific antibody molecule comprises two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain; (A1) each of the VL chains is an antigen-binding site for MDR1, having the following amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX 2 TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) wherein X 2 is N, Q, or S; The first VH chain is an antigen-binding site for MDR1, having the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQTPEKRLEWVATISSGGGX 1 TYYPDSVKGRFTVSRDNAMSSLYLQMSSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVS (SEQ ID NO: 418) wherein X 1 is N, Q, or S; the second VH chain is an antigen-binding site for ABCG2, having the following amino acid sequence: EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQTPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVSA (SEQ ID NO: 13) an antigen-binding site comprising HCDR1-3 of a VH chain having the second VH chain binds to ABCG2 when paired with one of the light chains; or (A2) each of the VL chains is an antigen-binding site for MDR1, having the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367) an antigen-binding site comprising LCDR1-3 of a VL chain having The first VH chain is an antigen-binding site for MDR1, having the following amino acid sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTF SRYTMSWVRQTPEKRLEWVATISSGGGX １ TYYPDSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVS (SEQ ID NO: 418) and HCDR1-3 of a VH chain having the formula: １ is N, Q, or S; the second VH chain is an antigen-binding site for ABCG2, having the following amino acid sequence: EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQTPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVSA (SEQ ID NO: 13) an antigen-binding site comprising HCDR1-3 of a VH chain having the second VH chain binds to ABCG2 when paired with one of the light chains; or (C1) each of the VL chains is an antigen-binding site for ABCG2, having the following amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDVNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVESEDLGIYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 199) an antigen-binding site comprising LCDR1-3 of a VL chain having the first VH chain is an antigen-binding site against ABCG2, having the following amino acid sequence: QVQLQQSGADLVRPGASVKLSCTASGFNIKDDYVHWVKQRPEQGLEWIGRIDPANGNTRYAPKFRGKATMTADTSSNTAYLQLSSLTSADTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 16) an antigen-binding site comprising HCDR1-3 of a VH chain having the second VH chain comprises an antigen-binding site for the TAA, the second VH chain comprising the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 388) wherein the TAA is EGFR; the second VH chain binds to a TAA when paired with one of the light chains; or (C2) each of the VL chains has an antigen-binding site for ABCG2, the antigen-binding site having the following amino acid sequence: DIQMTQSSSYLSVSVGGRVTITCKASDQINYWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSFQTEDVATYYCQQYWTTPYTFGGGTKVEIK (SEQ ID NO: 198) an antigen-binding site comprising LCDR1-3 of a VL chain having the first VH chain is an antigen-binding site against ABCG2, having the following amino acid sequence: QGQMHQSGAELVKPGASVKLSCKTSGFTFNSGYISWLKQKPRQSLEWIAWIYAGTGISNFNQKFTGKAQLTVDTSSSTAYMQLSSLTSADSAIYFCASGARKTLDFWGQGTSVTVSS (SEQ ID NO: 15) an antigen-binding site comprising HCDR1-3 of a VH chain having the second VH chain comprises an antigen-binding site for the TAA, the second VH chain comprising the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 388) wherein the TAA is EGFR; the second VH chain binds to a TAA when paired with one of the light chains; or (C3) each of the VL chains is an antigen-binding site for ABCG2, having the following amino acid sequence: DIVMTQSPSLLSASVGDRVTLSCKAGQNINNYLAWYQQKLGEAPKLLIYNANSLQTGIPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYNSWTTFGSGTKLEIK (SEQ ID NO: 206) an antigen-binding site comprising LCDR1-3 of a VL chain having the first VH chain is an antigen-binding site against ABCG2, having the following amino acid sequence: EVKLLESGPGLVKPSQSLSLTCSVTGYTITSGYDWSWIRKFPGNKMEWMGYISYSGWTNYNPSLRSRISISRDTSKNQFFLQLNSVTTEDTATYYCARVRGYNPFAYWGQGTLVTVSS (SEQ ID NO: 25) an antigen-binding site comprising HCDR1-3 of a VH chain having the second VH chain comprises an antigen-binding site for the TAA, the second VH chain comprising the following amino acid sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 388) wherein the TAA is EGFR; the second VH chain binds to a TAA when paired with one of the light chains; or (C4) Each of the VL chains has an antigen-binding site for ABCG2, the amino acid sequence being: DIVMTQSPSLLSASVGDRVTLSCKAGQNINNYLAWYQQKLGEAPKLLIYNANSLQTGIPSRFSGSGSGTDFTLTISSLQPEDVATYFCQQYNSWTTFGSGTKLEIK (SEQ ID NO: 206) an antigen-binding site comprising LCDR1-3 of a VL chain having the first VH chain is an antigen-binding site against ABCG2, having the following amino acid sequence: EVKLLESGPGLVKPSQSLSLTCSVTGYTITSGYDWSWIRKFPGNKMEWMGYISYSGWTNYNPSLRSRISISRDTSKNQFFLQLNSVTTEDTATYYCARVRGYNPFAYWGQGTLVTVSS (SEQ ID NO: 25) an antigen-binding site comprising HCDR1-3 of a VH chain having the second VH chain comprises an antigen-binding site for the TAA, the second VH chain comprising the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSD SWIHWVRQAPGKGLEWVAWISPYGGSTY YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS wherein the TAA is PD-L1; wherein said second VH chain binds to a TAA when paired with one of said light chains; wherein the antibody binds to cancer cells that express both ABCG2 and (i) a TAA or (ii) MDR1, but exhibits reduced binding to non-cancer cells that express only ABCG2 and either (i) a TAA or (ii) MDR1; wherein the CDRs are those defined by Kabat, Chothia, or MacCallum. Bispecific antibody molecule.

2. Corresponding to (A1) or (A2) above, (a) the first VH chain comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 418; or the first VH chain comprises an HCDR1 comprising the sequence GFTFSRYTMS (SEQ ID NO:419), an HCDR2 comprising the sequence VATISSGGGNTYYPDSVKG (SEQ ID NO:362), VATISSGGGQTYYPDSVKG (SEQ ID NO:363), or VATISSGGGSTYYPDSVKG (SEQ ID NO:364), and an HCDR3 comprising the sequence ARYGAGDAWFAY (SEQ ID NO:365); and / or (a) the second VH chain comprises an HCDR1 comprising the sequence: NNAMS (SEQ ID NO: 82), an HCDR2 comprising the sequence: TITGGGSYTYYPDSVKG (SEQ ID NO: 112), and an HCDR3 comprising the sequence: PDGNYEGVLAY (SEQ ID NO: 154); and / or the second VH chain comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence: EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQTPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVSA (SEQ ID NO: 13); and / or (C-1) The two identical VL chains comprise light chain CDR1-3 (LCDR1-3) of the VL chain of an anti-MDR1 antibody having the amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367), and the two identical VL chains (i) an LCDR1 comprising the sequence: RSSQSIVHSTGNTYLE (SEQ ID NO: 368); and (ii) an LCDR2 comprising the sequence: KVSNRFS (SEQ ID NO: 305); and (iii) an LCDR3 comprising the sequence: QGSHFPRT (SEQ ID NO: 369); or the VL chain comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% identity to the amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367); or (C-2) The two identical VL chains have the amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX ２ TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) (wherein X ２ is N, Q, or S), and The VL chain has the amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX ２ TYLEWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) (wherein X ２ is N, Q, or S), or the two identical VL chains (i) Sequence: RSSQSIVHSTGX ２ LCDR1 comprising TYLE (SEQ ID NO: 371); (ii) an LCDR2 comprising the sequence: KISNRFS (SEQ ID NO: 372); and (iii) an LCDR3 comprising the sequence: FQASHFPRT (SEQ ID NO: 373), wherein X ２ is N, Q, or S; The bispecific antibody molecule of claim 1.

3. Corresponding to (C1), (C2), (C3), or (C4) above, The first VH chain is selected from the group consisting of:

2. The bispecific antibody molecule of claim 1, comprising heavy chain complementarity determining regions 1 to 3 (HCDR1 to 3) of the anti-ABCG2 antibody described in .

4. The antigen-binding sites of the two VL chains are as shown in the following table:

4. The bispecific antibody molecule of claim 3, comprising the light chain CDRs 1-3 (LCDRs 1-3) of the antibody described in

5. (A) the first VH chain has the amino acid sequence: QVQLQQSGADLVRPGASVKLSCTASGFNIKDDYVHWVKQRPEQGLEWIGRIDPANGNTRYAPKFRGKATMTADTSSNTAYLQLSSLTSADTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 16), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPANGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 17), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPAQGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 18), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQAPGQGLEWIGRIDPASGNTRYAPKFRGRATMTADTSISTAYMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS (SEQ ID NO: 19) and / or comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to (a) the VL chain has the amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDVNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVESEDLGIYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 199), or DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDVNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQTTHVPYTFGGGTKLEIK (SEQ ID NO: 200) comprising an amino acid sequence having at least 90%, at least 95%, or 100% identity to A bispecific antibody molecule described in (C1) of claim 1.

6. (a) the first VH chain comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence: QGQMHQSGAELVKPGASVKLSCKTSGFTFNSGYISWLKQKPRQSLEWIAWIYAGTGISNFNQKFTGKAQLTVDTSSSTAYMQLSSLTSADSAIYFCASGARKTLDFWGQGTSVTVSS (SEQ ID NO: 15); and / or (a) the VL chain comprises an amino acid sequence having at least 90%, at least 95%, or 100% identity to the amino acid sequence: DIQMTQSSSYLSVSVGGRVTITCKASDQINYWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSFQTEDVATYYCQQYWTTPYTFGGGTKVEIK (SEQ ID NO: 198); A bispecific antibody molecule described in (C2) of claim 1.

7. The table below: a first VH chain comprising HCDR1-3 as shown in Figure 1, a second VH chain comprising HCDR1-3 as shown in Figure 1, and a common VL chain comprising LCDR1-3.

8. 1. A pharmaceutical composition comprising: A bispecific antibody molecule according to any one of claims 1 to 7, and a pharmaceutically acceptable excipient.

9. further comprising an additional active agent; (i) the additional active agent is a chemotherapeutic agent; (ii) the additional active agent is an inhibitor of a multidrug resistance transporter; or (iii) the additional active agent is an immunotherapeutic agent; The pharmaceutical composition of claim 8.

10. 10. A pharmaceutical composition comprising the bispecific antibody molecule of any one of claims 1 to 7 for use in a method for treating cancer in a subject, said method comprising administering to said subject a therapeutically effective amount of said bispecific antibody molecule.

11. One or more nucleic acids comprising one or more sequences encoding the bispecific antibody molecule of any one of claims 1 to 7.

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

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

14. An immune effector cell comprising a chimeric antigen receptor (CAR) comprising an ABCG2-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the ABCG2-binding domain comprises heavy chain complementarity-determining regions 1 to 3 (HCDR1 to 3) and light chain CDR1 to 3 (LCDR1 to 3) of a pair of variable heavy chain (VH) and variable light chain (VL) regions of a bispecific antibody molecule according to any one of claims 1 to 7.

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