ABCG2 efflux pump-cancer antigen multispecific antibodies and related compositions, reagents, kits, and methods

Abstract

Multispecific antibodies that target both the cellular efflux pump ABCG2 and a cancer-associated antigen are provided, as well as pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits that contain or encode such multispecific antibodies. Methods of treating a subject for cancer are also provided, comprising administering to the subject a multispecific antibody that targets both a cellular efflux pump and a cancer-associated antigen. Methods of producing the described multispecific antibodies and their related reagents are also provided, including genetically modified cell lines useful in the methods and methods of making such genetically modified cell lines.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 034,822, 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-005WO SEQ LIST_ST25.txt" having a size of 87 KB, created on May 27, 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 the cell. 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 (ABCG2), 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] Among cancer patients, when metastatic cancer cell populations have been largely killed and eliminated from the patient through the use of chemotherapy, it is not uncommon for drug-resistant cancer cell populations to emerge and spread, becoming unresponsive to retreatment with the previous therapy. In most cases, different drugs with different mechanisms of action are applied until another emerging drug-resistant cell population and / or tumor develops.

[0007] Therefore, there is a need to develop reagents that can be used to inhibit EP. Summary of the Invention

[0008] Anti-ABCG2 antibodies that can be used as multispecific antibodies targeting both ABCG2 and tumor-associated antigens (TAA) are provided, as well as pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits that contain or encode such multispecific antibodies. The multispecific antibodies comprise a common variable light (VL) chain containing an antigen-binding site for ABCG2, a first variable heavy (VH) chain containing an antigen-binding site for ABCG2, and a second VH chain containing an antigen-binding site for a TAA, or comprise a first VH chain and a first VL chain containing an antigen-binding site for ABCG2, and a second VH chain and a second VL chain containing an antigen-binding site for a TAA. Also provided are methods for treating a subject for cancer, comprising administering a multispecific antibody that targets both ABCG2 and a TAA to the subject. Treating can involve administering the multispecific antibody alone, or administering the multispecific antibody and a chemotherapeutic agent. Additionally, methods for producing the described multispecific antibodies and their related reagents are provided, including genetically modified cell lines useful in the subject methods and methods for making such genetically modified cell lines.

[0009] The bispecific antibodies provided herein bind to cancer cells that express both ABCG2 and a TAA, but exhibit reduced binding to non-cancer cells that express ABCG2 and / or a TAA. In other words, the bispecific antibodies provided herein bind with low affinity to (1) cells that express a TAA but have low or no ABCG2 expression, and (2) cells that express ABCG2 but have low or no TAA expression, and bind with high affinity to cancer cells that express at least one or both of ABCG2 and a TAA at relatively high levels, i.e., at levels higher than normal cells. [Brief explanation of the drawings]

[0010] [Figure 1]Chemosensitivity analyses are provided showing that 293T cells overexpressing ABCG2 have increased sensitivity to topotecan in the presence of the anti-ABCG2 antibody 5D3 and a bispecific antibody that binds to ABCG2 and CD47. The bispecific antibody 5D3DDKT14KK5D3 contains the heavy chain (HC) and light chain (LC) from the anti-ABCG2 antibody 5D3 and the HC from the anti-CD47 antibody 5F9. The bispecific antibody 5D3hVHv1DDKT14KK5D3hVLv1 contains the HC and LC from the anti-ABCG2 antibody 5D3 and the HC from the anti-CD47 antibody 5F9. The bispecific antibody 5D3hVHv2DDKT14KK5D3hVLv1 contains the HC and LC from the anti-ABCG2 antibody 5D3 and the HC from the anti-CD47 antibody 5F9. hVHv1 and hVHv2 refer to the humanized variable heavy chain types 1 and 2 of 5D3 HC, respectively. hVLv1 refers to the humanized variable light chain type 1 of 5D3 LC. KT14 refers to CD47. DD and KK refer to charge pair substitutions that enhance pairing between 5D3 HC and 5F9 HC. IC50 values ​​are in nM. [Figure 2] Binding of 5D3 and humanized 5D3 antibodies to 293T-G2OX cells is shown along with the corresponding EC50 binding affinities. EC50 values ​​are in nM. [Figure 3] Figure 1 shows the binding of the humanized ABCG2 / KT9 bispecific antibody to 3T3 cells stably transfected to express ABCG2 (3T3-G2), 293T cells stably transfected to express human ABCG2 (293T_ABCG2_OX), and 293T cells stably transfected to express human KT9 (293T-KT9OX). KT9 is the anti-PD-L1 antibody atezolizumab, and 5D3 is the anti-ABCG2 antibody. The humanized bispecific antibodies 5D3hVH-v1DD KT9KK 5D3hVL-v1 and 5D3hVH-v2DD KT9KK 5D3hVL-v1 contain the HC and LC from the anti-ABCG2 antibody 5D3 and the HC from the anti-PD-L1 antibody KT9. [Figure 4]Binding of the humanized 5D3 / KT9 / 5D3 bispecific antibody to 293T cells stably transfected with ABCG2 (293T-G2OX), KT9 (293T-KT9OX), and both ABCG2 and KT9 (293T-G2KT9OX) is shown, along with the corresponding EC50 binding affinity values. [Figure 5] Figure 1 shows the results of a xenograft study in which the cytotoxic activity of an ABCG2 / PD-L1 bispecific antibody (5D3 / KT9), alone or in combination with topotecan, was tested in the HT1376 (ATCC, CRL-1472) human bladder epithelial carcinoma cell line. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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, and Fd fragments, chimeric antibodies, humanized 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 may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, or the like. Antibodies may also 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 may also be bound to solid supports, including, but not limited to, polystyrene plates or beads. The terms also encompass Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. Antibodies may be monovalent or bivalent. The antibodies used herein can be used to assay the expression of a target antigen on the cell surface, for example, in a cell or tissue sample from a patient.

[0012] 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.

[0013] "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 of each variable domain interact to form the V H -V L The six CDRs define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can form an antigen-binding site that has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site containing the three CDRs of each variable domain.

[0014] 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 pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0015] 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.

[0016] "Single-chain Fv," "sFv," or "scFv" antibody fragments are fragments of the V of an antibody. H and V L In 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).

[0017] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) on the same polypeptide chain (V H -V L) 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 the complementary domains on another chain and 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).

[0018] 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.

[0019] The term "binding" refers to a direct association between two molecules through 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. Non-specific binding occurs at approximately 10 -7Binding 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.

[0020] 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 described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and 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 which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. [Table 1]

[0021] 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.

[0022] 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.

[0023] As used herein, the term "immunoglobulin" refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include 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, the subject antibody comprises a whole immunoglobulin, including a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.

[0024] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to an antigen. Examples of binding fragments include: (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).

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

[0026] 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.

[0027] 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.

[0028] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human framework regions (FRs). At least a portion of the humanized antibody constant region is derived from a human antibody. In preferred embodiments of the antibody molecules disclosed herein, the constant region is from a human IgG antibody, such as human IgG1. In preferred embodiments, the antibody molecules disclosed herein comprise 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 preferred embodiments, the antibody molecules disclosed herein comprise a light chain comprising a variable light chain region provided herein and a human light chain constant region. In preferred embodiments, the human light chain constant region is a human kappa light chain constant region. In certain aspects, the human IgG1 heavy chain constant region present in the 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.

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

[0030] The term "epitope" refers to a 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] The percent identity between a pair of sequences can be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region, including gaps. 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).

[0039] The phrase "conservative amino acid substitution" refers to substitutions of amino acid residues within the following groups: 1) L, I, M, V, F, 2) R, K, 3) F, Y, H, W, R, 4) G, A, T, S, 5) Q, N, 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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).

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

[0046] "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.

[0047] 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.

[0048] 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.

[0049] Anti-ABCG2 antibodies that can be used as multispecific antibodies targeting both ABCG2 and tumor-associated antigens (TAA) are provided, as well as pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such multispecific antibodies. The multispecific antibodies comprise a common variable light (VL) chain containing an antigen-binding site for ABCG2, a first variable heavy (VH) chain containing an antigen-binding site for ABCG2, and a second VH chain containing an antigen-binding site for a TAA, or comprise a first VH chain and a first VL chain containing an antigen-binding site for ABCG2, and a second VH chain and a second VL chain containing an antigen-binding site for a TAA, where the first and second VL chains are different. Also provided are methods for treating a subject for cancer, comprising administering a multispecific antibody targeting both ABCG2 and a TAA to the subject. Treating can involve administering the multispecific antibody alone, or administering the multispecific antibody and a chemotherapeutic agent. Additionally, methods for producing the described multispecific antibodies and their related reagents are provided, including genetically modified cell lines useful in the subject methods and methods for making such genetically modified cell lines.

[0050] The bispecific antibodies provided herein bind to cancer cells that express both ABCG2 and a TAA, but exhibit reduced binding to non-cancer cells that express ABCG2 and / or a TAA. In other words, the bispecific antibodies provided herein bind with low affinity to (1) cells that express a TAA but have low or no ABCG2 expression, and (2) cells that express ABCG2 but have low or no TAA expression, and bind with high affinity to cancer cells that express at least one or both of ABCG2 and a TAA at relatively high levels, i.e., at levels higher than normal cells.

[0051] 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.

[0052] 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.

[0053] 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 near the number preceded by the term. When determining whether a number is near or approximately near a specifically recited number, the near or approximately near, but unrecited number may be a number that, in the context in which it is presented, provides substantially the same value as the specifically recited number.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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).

[0059] antibody bispecific antibody The present disclosure provides a bispecific antibody molecule that binds to multidrug resistance protein 1 (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, each of which comprises an antigen-binding site for ABCG2, the first VH chain comprising an antigen-binding site for ABCG2, and the second VH chain comprising an antigen-binding site for the TAA, which binds to the TAA when paired with one of the light chains; or comprising a first VH chain and a first VL chain comprising an antigen-binding site for ABCG2, and a second VH chain and a second VL chain comprising an antigen-binding site for the TAA, the first and second VL chains having different sequences. The bispecific antibody binds to cancer cells expressing both ABCG2 and the TAA, but exhibits reduced binding to non-cancer cells expressing ABCG2 and / or the TAA. In other words, the bispecific antibodies provided herein bind with low affinity to (1) cells that express a TAA but have low or no ABCG2 expression, and (2) cells that express ABCG2 but have low or no TAA expression, and bind with high affinity to cancer cells that express at least one or both of ABCG2 and a TAA at relatively high levels, i.e., at levels higher than normal cells.

[0060] A relatively high level refers to expression that is at least 1.5 times (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more) the expression level in normal cells of the same type as the cancer cells. Reduced affinity refers to binding affinity that is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, or more) compared to the binding of the antibody molecule to normal cells of the same type as the cancer cells. Reduced affinity also encompasses the absence of detectable binding.

[0061] 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 derived 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.

[0062] In certain embodiments, the antigen binding sites of the two VL chains have the sequences: It contains the VL chain light chain CDRs (LCDR1 to 3) having the sequence DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1).

[0063] The VL chain amino acid sequence shown in SEQ ID NO: 1 is the sequence of the VL chain of the anti-ABCG2 antibody 5D3.

[0064] In certain embodiments, the CDRs of the VL and VH light chains may be defined based on the Kabat nomenclature.

[0065] In certain embodiments, i) LCDR1 comprises the sequence: KASGYILNRLA (SEQ ID NO: 2), (ii) LCDR2 comprises the sequence: GATSLET (SEQ ID NO: 3), and (iii) LCDR3 comprises the sequence: QQYWSTPWT (SEQ ID NO: 4). These LCDRs are based on the Kabat nomenclature.

[0066] In certain embodiments, the two VL chains are humanized. In certain embodiments, the two VL chains are humanized to include framework regions from a human antibody.

[0067] In certain embodiments, the two VL chains have the sequences: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1).

[0068] In certain embodiments, the bispecific antibody comprises a light chain comprising a VL chain and a light chain constant region described herein. The light chain constant region may be a human immunoglobulin kappa chain constant region having the amino acid sequence set forth in UniProtKB / Swiss-Prot:P01834.2.

[0069] In certain embodiments, the bispecific antibody molecule comprises a first VH chain, wherein the VH chain has the sequence: It contains heavy chain CDRs 1 to 3 (HCDRs 1 to 3) of a VH chain having the sequence QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5).

[0070] The VH chain amino acid sequence shown in SEQ ID NO: 5 is the sequence of the VH chain of the anti-ABCG2 antibody 5D3.

[0071] In certain embodiments, HCDRs 1-3 of the VH chain are defined according to the Kabat nomenclature.

[0072] In a particular embodiment, the first VH chain comprises (i) an HCDR1 comprising the sequence SDYAWN (SEQ ID NO: 63), (ii) an HCDR2 comprising the sequence YINFDGGTTYNPSLRG (SEQ ID NO: 64), and iii) an HCDR3 comprising the sequence FYGAKGTLDY (SEQ ID NO: 65). These HCDRs are based on the Kabat nomenclature.

[0073] In certain embodiments, the first and / or second VH chain is humanized. In certain embodiments, the VH chain is humanized to include framework regions from a human antibody.

[0074] In certain embodiments, the first VH chain has the sequence: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5), EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWMGYINFDGGTTYNPSLRGRITISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 6), or EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWIGYINFDGGTTYNPSLRGRVTISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 7).

[0075] In certain embodiments, the first VH chain comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0076] In certain embodiments, the bispecific antibody comprises a first VH chain described herein and a first heavy chain comprising a human IgG1 heavy chain constant region.

[0077] In certain embodiments, the second VH chain of the bispecific antibody is derived from a monospecific antibody molecule that binds to a TAA, and the affinity of the bispecific antibody molecule for the TAA is at least two-fold lower (e.g., at least three-fold lower, at least four-fold lower, at least five-fold lower) than the affinity of the monospecific antibody molecule for the TAA from which the VH chain is derived. The affinities of the bispecific antibody and the monospecific antibody are measured using the same assay. Any suitable method for measuring antibody affinity may be utilized. In certain embodiments, affinity may be measured by calculating the equilibrium constant for the reversible binding of an antibody to an antigen, expressed as the dissociation constant (Kd). In certain embodiments, Kd may be measured by ELISA.

[0078] In certain embodiments, the second VH chain of the bispecific antibody is derived from a monospecific antibody molecule that binds to a TAA, and the half-maximal effective concentration (EC50) of the bispecific antibody molecule for the TAA is at least two-fold higher (e.g., at least three-fold higher, at least four-fold higher, at least five-fold higher) than the EC50 of the monospecific antibody molecule for the TAA from which the VH chain is derived. The EC50 of the bispecific antibody and the monospecific antibody are measured using the same assay. Any suitable method for measuring the EC50 of an antibody can be utilized. The concentration providing half of the maximal response (e.g., half of the maximal fluorescence intensity) is measured as the EC50.

[0079] The EC50 of a test antibody can be determined by flow cytometry or ELISA. For example, flow cytometry can involve contacting cells expressing an antigen (e.g., human wild-type ABCG2 or mutant ABCG2) with the antibody (serialized dilutions of the antibody) 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 can 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 can be removed and the cells can be washed. The washed cells can be sorted by flow cytometry, and the number of cells bound to the fluorescently labeled secondary antibody can be counted. The concentration 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 can be 293T cells overexpressing ABCG2.

[0080] A TAA can be any antigen known to be overexpressed in cancer cells. 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. For example, a TAA can be a neoantigen, a class of tumor antigens that arise from tumor-specific mutations that alter the amino acid sequence of the encoded protein compared to the amino acid sequence of the unmutated protein. In other embodiments, a TAA is an antigen that is expressed in normal cells but at a higher level in cancer cells. In certain embodiments, a TAA can be CD47, ErbB1, ErbB2, or PD-L1.

[0081] Anti-ABCG2 and anti-CD47 bispecific antibody In certain embodiments, the bispecific antibody molecule binds to CD47 and the second VH chain has the amino acid sequence: It contains the HCDR of the VH chain comprising QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 8).

[0082] The VH chain amino acid sequence shown in SEQ ID NO: 8 is the sequence of the VH chain of the anti-CD47 antibody 5F9.

[0083] In a specific embodiment, the second VH chain comprises HCDR1 comprising the sequence NYNMH (SEQ ID NO: 9), HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD (SEQ ID NO: 10), and HCDR3 comprising the sequence GGYRAMDY (SEQ ID NO: 11). HCDRs 1-3 are defined according to the Kabat nomenclature.

[0084] In certain embodiments, the second VH chain comprises a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:8.

[0085] In certain embodiments, the bispecific antibody comprises a first VH chain and a first VL chain, and a second VH chain and a second VL chain, wherein the first VH chain and VL chain bind to ABCG2 and the second VH chain and VL chain bind to CD47. The first VH chain and VL chain may be as described in the preceding section, the common light chain may be the first VL chain, the second VH chain may be as described above, and the second VL chain may be a VL chain comprising LCDR1-3 of the VL chain of an anti-CD47 antibody. In a specific embodiment, the bispecific antibody comprises a first VH chain comprising HCDR1 to 3 having the amino acid sequences set forth in SEQ ID NOs: 63 to 65, respectively; a first VL chain comprising LCDR1 to 3 having the amino acid sequences set forth in SEQ ID NOs: 2 to 4, respectively; a second VH chain comprising HCDR1 to 3 having the amino acid sequences set forth in SEQ ID NOs: 9 to 11, respectively; and a second VL chain comprising LCDR1 to 3 of a VL chain having the amino acid sequence set forth in SEQ ID NO: 60.

[0086] Additional embodiments of bispecific antibodies are described elsewhere in this application and include humanized versions of the sequences disclosed herein and / or substitutions in the Fc region that promote heterodimer formation between a first VH chain and a second VH chain.

[0087] Anti-ABCG2 and anti-ErbB2 bispecific antibody In certain embodiments, the TAA can be ErbB2. ErbB2 is also known as receptor tyrosine kinase 2 or HER2. In certain embodiments, the bispecific antibody molecule that binds to ABCG2 and ErbB2 comprises a common light chain and a first VH chain as described in the preceding section, and the second VH chain has the amino acid sequence: It contains HCDRs 1 to 3 of the VH chain of mAb pertuzumab, which contains EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 12).

[0088] HCDRs 1 to 3 defined according to the Kabat nomenclature are as follows: HCDR1: DYTMD (SEQ ID NO: 13) HCDR2: DVNPNSGGSIYNQRFKG (SEQ ID NO: 14) HCDR3: NLGPSFYFDY (SEQ ID NO: 15)

[0089] The second VH chain of the bispecific antibody that binds ABCG2 and ErbB2 may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:12.

[0090] The second VH chain of the bispecific antibody that binds to ABCG2 and ErbB2 has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 16).

[0091] As described elsewhere herein, at least one, two, or all of the VL chain, first VH chain, and second VH chain may be humanized. In addition, the Fc region of the VH chain may contain substitutions that increase heterodimerization between the first VH chain and the second VH chain. In certain embodiments, the first heavy chain may be humanized and contain charge-pair substitutions K392D and K409D, and the second heavy chain contains charge-pair substitutions E356K and D399K.

[0092] In certain embodiments, the bispecific antibody comprises a first VH chain and a first VL chain, and a second VH chain and a second VL chain, wherein the first VH chain and VL chain bind to ABCG2 and the second VH chain and VL chain bind to HER2. The first VH chain and VL chain may be as described in the preceding section, the common light chain is the first VL chain, the second VH chain is as described above, and the second VL chain may be a VL chain comprising LCDR1-3 of the VL chain of the anti-HER2 antibody.

[0093] Anti-ABCG2 and anti-EGFR bispecific antibody In a specific embodiment, the TAA can be epidermal growth factor receptor (EGFR). EGFR is also known as the receptor tyrosine-protein kinase ErbB1 and HER1. HCDRs 1-3 in the second VH chain, which contain the antigen-binding site for EGFR, can be derived from the VH chain of the anti-EGFR antibody necitumumab or cetuximab. The heavy chain of necitumumab has the following sequence: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQG TLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)

[0094] The heavy chain of cetuximab has the following sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18)

[0095] In a first embodiment, the anti-ABCG2 anti-EGFR bispecific antibody comprises a VL and a first VH chain as described in the preceding section, and the second VH chain may comprise an HCDR from the VH region of necitumumab. The HCDR, defined according to the Kabat nomenclature, may have the following sequence: HCDR1: SGDYYWS (SEQ ID NO: 19) HCDR2: YIYYSGSTDYNPSLKS (SEQ ID NO: 20) HCDR3: VSIFGVGTFDY (SEQ ID NO: 21)

[0096] In certain embodiments, the second VH chain has the amino acid sequence: It may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 22).

[0097] The second VH chain of a bispecific antibody that binds ABCG2 and EGFR can be present in a heavy chain having an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:17.

[0098] In a second embodiment, the anti-ABCG2 anti-EGFR bispecific antibody comprises a VL and a first VH chain as described in the preceding section, and the second VH chain may comprise an HCDR from the VH region of cetuximab. The HCDR, defined according to the Kabat nomenclature, may have the following sequence: HCDR1: NYGVH (SEQ ID NO: 23) HCDR2: VIWSGGNTDYNTPFTS (SEQ ID NO: 24) HCDR3: ALTYYDYEFAY (SEQ ID NO: 25)

[0099] In certain embodiments, the second VH chain has the amino acid sequence: It may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 26).

[0100] The second VH chain of a bispecific antibody that binds ABCG2 and EGFR can be present in a heavy chain having an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:18.

[0101] In a specific embodiment, the bispecific antibody comprises a first VH chain and a first VL chain, and a second VH chain and a second VL chain, wherein the first VH chain and VL chain bind to ABCG2 and the second VH chain and VL chain bind to EGFR. The first VH chain and VL chain may be as described in the preceding section, the common light chain may be the first VL chain, the second VH chain may be as described above, and the second VL chain may be a VL chain comprising LCDR1-3 of the VL chain of an anti-EGFR antibody.

[0102] Anti-ABCG2 and anti-PD-L1 bispecific antibody In certain embodiments, the TAA can be programmed death-ligand 1 (PD-L1). PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). In certain embodiments, the bispecific antibody molecule that binds to ABCG2 and PD-L1 comprises a common light chain and a first VH chain as described in the preceding section, and the second VH chain has the amino acid sequence: It comprises HCDRs 1 to 3 of a VH chain comprising EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 27).

[0103] HCDRs 1 to 3 defined according to the Kabat nomenclature are as follows: HCDR1: DSWIH (SEQ ID NO: 28) HCDR2: WISPYGGSTYYADSVKG (SEQ ID NO: 29) HCDR3: RHWPGGFDY (SEQ ID NO: 30)

[0104] The second VH chain of a bispecific antibody that binds ABCG2 and PD-L1 may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:27.

[0105] The second VH chain of the bispecific antibody that binds ABCG2 and PD-L1 has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31).

[0106] In certain embodiments, the bispecific antibody comprises a first VH chain and a first VL chain, and a second VH chain and a second VL chain, where the first VH chain and VL chain bind to ABCG2 and the second VH chain and VL chain bind to PD-L1. The first VH chain and VL chain may be as described in the preceding section, the common light chain is the first VL chain, the second VH chain is as described above, and the second VL chain may be a VL chain comprising LCDR1-3 of the VL chain of the anti-PD-L1 antibody.

[0107] As described elsewhere herein, at least one, two, or all of the VL chain (or the first and second VL chains), the first VH chain, and the second VH chain may be humanized. In addition, the Fc region of the VH chain may contain substitutions that increase heterodimerization between the first VH chain and the second VH chain. In certain embodiments, the first heavy chain may be humanized.

[0108] The first HC may comprise charge pair substitutions K392D and K409D and the second heavy chain may comprise charge pair substitutions E356K and D399K, or vice versa.

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

[0110] In certain embodiments, the bispecific antibody molecule specifically binds to cells expressing both ABCG2 and a TAA, with greater than two-fold affinity for cells expressing both ABCG2 and a TAA compared to cells expressing either ABCG2 or a TAA.

[0111] In certain embodiments, the bispecific antibody molecule inhibits ABCG2-mediated efflux when bound to cells expressing ABCG2. Inhibition can be a decrease in efflux by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or more, compared to efflux by ABCG2 in the absence of the bispecific antibody.

[0112] In certain embodiments, the bispecific antibody molecule comprises an Fc domain that has been modified to reduce or abolish binding of the antibody to one or more Fcγ receptors, hi certain embodiments, the IgG1 Fc domain may have one or more of the following substitutions: L234A, L235A, P329G, and N297A / Q / G.

[0113] As summarized above, the present disclosure provides multispecific antibodies having a domain that targets a cellular efflux pump and a domain that targets a cancer-associated antigen. Included are multispecific antibodies that contain a multidrug resistance protein 1 (ABCG2)-binding domain and a leukocyte surface antigen CD47-binding domain. Such multispecific antibodies of the present disclosure specifically bind to cells that express both ABCG2 and CD47.

[0114] In one embodiment, the multispecific antibody of the present disclosure targets both ABCG2 and CD47. 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 and responsible for reduced drug accumulation in multidrug-resistant cells. CD47, also known as integrin-associated protein (IAP), is an immunoglobulin superfamily transmembrane protein that binds to membrane integrins and also functions as a receptor for the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα), and is encoded by the CD47 gene. CD47 ligand binding can result in the inhibition of phagocytosis; therefore, as a target in immunotherapy, masking the CD47 extracellular domain prevents the inhibition of immune-mediated killing of CD47-expressing cancer cells.

[0115] The multispecific antibodies of the present disclosure comprise an ABCG2-binding domain and a CD47-binding domain, and optionally all or part of an Fc domain. In the presence of cells that express ABCG2 but have low or no CD47 expression, the multispecific antibodies have low affinity for the cells. Correspondingly, in the presence of cells that express CD47 but have low or no ABCG2 expression, the multispecific antibodies have low affinity for the cells. However, in the presence of cells that express both ABCG2 and CD47, the multispecific antibodies have high affinity for the cells.

[0116] Thus, multispecific antibodies of the present disclosure bind with higher affinity to cells expressing both ABCG2 and CD47 than to cells expressing only ABCG2 or CD47. Correspondingly, multispecific antibodies of the present disclosure bind with significantly reduced affinity when the corresponding secondary target is present at low levels, e.g., compared to when both the primary and secondary targets are present at above-low levels (e.g., average, normal, and / or high levels). In some embodiments, the affinity with which a subject multispecific antibody binds to cells expressing both ABCG2 and CD47 is more than 2-fold, including, for example, more than 2.5-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, more than 9-fold, more than 10-fold, or more, compared to the affinity with which a subject multispecific antibody binds to cells expressing either ABCG2 or CD47 (or low levels of either ABCG2 or CD47).

[0117] In some embodiments, a subject multispecific antibody may interfere with the function of cellular ABCG2 protein when bound to a cell expressing ABCG2. Thus, a multispecific antibody of the present disclosure may inhibit efflux by ABCG2 protein, e.g., efflux is reduced by 5% or more, including 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 a subject multispecific antibody.

[0118] In some embodiments, a subject multispecific antibody, when bound to a cell expressing CD47, may interfere with the function of cellular CD47 protein. Thus, a multispecific antibody of the present disclosure may inhibit binding of a CD47 ligand or CD47 binding partner to CD47, e.g., ligand / binding partner binding is reduced by 5% or more, including 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, compared to binding by CD47 in the absence of a subject multispecific antibody.

[0119] The multispecific antibodies of the present disclosure are at least bispecific for ABCG2 and CD47, and the three-dimensional structure of the antibody may vary. The term "antibody" refers to a protein comprising one or more (e.g., one or two) heavy chain variable regions (VH) and / or one or more (e.g., one or two) light chain variable regions (VL), or subfragments thereof, capable of binding to an epitope. With respect to the multispecific antibodies described herein, such antibodies are capable of binding to at least two different epitopes present on two different target proteins. The number of different target proteins, and therefore the number of different epitopes, bound by a subject multispecific antibody may vary and may be two (i.e., bispecific), three (trispecific), four, or more.

[0120] In some embodiments, the multispecific antibodies of the present disclosure may comprise 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 binds to an ABCG2-specific heavy chain, and a second copy of the same light chain binds to a CD47-specific heavy chain.

[0121] The VH and VL regions can be further subdivided into regions of hypervariability called "complementarity-determining regions (CDRs)," interspersed with more conserved regions called "framework regions (FRs)." The extent of the FRs and CDRs has been precisely defined (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1987) J. Mol. Biol. 196: 901-917). The VH chain comprises 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.

[0122] The VH or VL chain of an antibody can further comprise all or a portion of a heavy or light chain constant region, thereby forming a heavy or light immunoglobulin chain, respectively. In one embodiment, an antibody is a tetramer of two heavy chains and two light chains, where the heavy and light chains are interconnected, for example, by disulfide bonds. The heavy chain constant region 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 an antibody 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, a subject antibody is an IgG isotype.

[0123] 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.

[0124] In some embodiments, the subject antibodies do not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, but instead comprise antigen-binding fragments of one or more full-length immunoglobulin heavy chains and / or one or more antigen-binding fragments of a full-length immunoglobulin light chain. In some embodiments, the antigen-binding fragments are comprised in separate polypeptide chains, while in other embodiments, the antigen-binding fragments are comprised within a single polypeptide chain.

[0125] The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that can specifically bind to ABCG2 or CD47 as described above. 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).

[0126] 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 may optionally comprise constant regions derived from human germline immunoglobulin sequences.

[0127] The modified antibodies may contain modified domains, including cases 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 procedures 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, when one heavy chain is modified to contain K392D and K409D substitutions and the other heavy chain is modified to contain E356K and D399K substitutions. Charge-pair substituted chains may preferentially form heterodimers with each other. The numbering of amino acid substitutions follows the EU numbering system of HC.

[0128] 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.

[0129] 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, J. B. et al. Protein Eng. 9(7):617-2 (1996); and Merchant, A. M. et al. Nat. Biotechnol. 16(7):677-81 (1998), the disclosures of which are incorporated herein in their entireties. In antibodies generated from paired knob-into-hole modified domains, bispecific heterodimers generally represent the majority.

[0130] As summarized above, the multispecific antibodies of the present disclosure comprise an ABCG2-binding domain and a CD47-binding domain. Such domains may vary, including the epitopes bound by the domains, variable region configurations and sequences, etc.

[0131] The subject ABCG2 binding domains specifically bind to one or more epitopes of ABCG2. Thus, the epitopes are ABCG2 epitopes. The size of the ABCG2 epitopes bound by the anti-ABCG2 binding domains can vary, including when the ABCG2 epitopes are formed by polypeptides having a continuous stretch of the ABCG2 sequence, which can range from 4 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.

[0132] In some embodiments, the ABCG2 epitope is, for example, the human ABCG2 sequence: (SEQ ID NO: 32), or its ECD1 (417-428): KNDSTGIQNRAG (SEQ ID NO: 33), its ECD2 (499-506): LKPKADAF (SEQ ID NO: 34), its ECD3 (557-630): NLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATGNNPCNYATCTGEEYLVKQGIDLSPWGLWKNH (SEQ ID NO: 35), or Mus musculus ABCG2 sequence: MSSSNDHVLVPMSQRNNNGLPRTNSRAVRTLAEGDVLSFHHITYRVKVKSGFLVRKTVEK EILSDINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPKGLSGDVLINGAPQPAHFKCCS GYVVQDDVVMGTLTVRENLQFSAALRLPTTMKNHEKNERINTIIKELGLEKVADSKVGTQ FIRGISGGERKRTSIGMELITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTIIFS IHQPRYSIFKLFDSLTLLASGKLVFHGPAQKALEYFASAGYHCEPYNNPADFFLDVINGD SSAVMLNREEQDNEANKTEEPSKGEKPVIENLSEFYINSAIYGETKAELDQLPGAQEKKG TSAFKEPVYVTSFCHQLRWIARRSFKNLLGNPQASVAQLIVTVILGLIIGAIYFDLKYDA AGMQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYISGYYRVSSYFFGKVMSDLLP MRFLPSVIFTCVLYFMLGLKKTVDAFFIMMFTLIMVAYTASSMALAIATGQSVVSVATLL MTIAFVFMMLFSGLLVNLRTIGPWLSWLQYFSIPRYGFTALQYNEFLGQEFCPGFNVTDN STCVNSYAICTGNEYLINQGIELSPWGLWKNHVALACMIIIFLTIAYLKLLFLKKYS (SEQ ID NO: 36), or its ECD1 (415 to 428): DLKYDAAGMQNRAG (SEQ ID NO: 37), its ECD2 (499 to 506): LKKTVDAF (SEQ ID NO: 38), its ECD3 (557 to 632): NLRTIGPWLSWLQYFSIPRYGFTALQYNEFLGQEFCPGFNVTDNSTCVNSYAICTGNEYLINQGIELSPWGLWKNH (SEQ ID NO: 39), non-human primate sequences, for example, Macaca fascicularis (cynomolgus monkey) sequence: MSSSNVEVFIPMSQENTNGFPTTTSNDRKAFTEGAVLSFHNICYRVKVKSGFLPGRKPVE KEILSNINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPSGLSGDVLINGALRPTNFCN SGYVVQDDVVMGTLTVRENLQFSAALRLPTTMTNHEKNERINRVIQELGLDKVADSKVGT QFIRGVSGGERKRTSIGMELITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTIIF SIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAYNNPADFFLDIING DSTAVALNREEDFKATEIIEPSKRDKPLVEKLAEIYVDSSFYKETKAELHQLSGGEKKKK ITWFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQIIVTVILGLVIGAIIFGLNNDS TGIQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYISGYYRVSSYFFGKLLSDLLP MRMLPSIIFTCIVYFMLGLKPTADAFFIMMFTLMMVAYSASSMALAIAAGQSVVSVATLL MTICFVFMMIFSGLLVNLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATVN NTCNYATCTGEEYLTKQGIDLSPWGLWKNHVALACMIVIFLTIAYLKLLFLKKYS (SEQ ID NO: 40), or its ECD1 (417 to 428): NNDSTGIQNRAG (SEQ ID NO: 41), its ECD2 (499 to 506): LKPTADAF (SEQ ID NO: 42), its ECD3 (557 to 630): NLTTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATVNNTCNYATCTGEEYLTKQGIDLSPWGLWKNH (SEQ ID NO: 43), or Pan troglodytesThe ABCG2 sequence 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 to a contiguous stretch of an ABCG2 sequence, including, but not limited to, the ABCG2 sequence: (SEQ ID NO: 44).

[0133] In some embodiments, the ABCG2 epitope may be formed by a mutated ABCG2 polypeptide. The mutated ABCG2 polypeptide may be derived from a human ABCG2 polypeptide. The human ABCG2 polypeptide may contain a mutation that results in an ABCG2 polypeptide having an open conformation. The mutant human ABCG2 polypeptide having an open conformation may 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 about 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:45), etc.

[0134] A subject ABCG2 binding domain exhibits high affinity binding to ABCG2. For example, a subject ABCG2 binding domain exhibits high affinity binding to ABCG2. -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10M, at least about 10 -11 M, or at least about 10 -12 M or 10 -12 The subject ABCG2 binding domain 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 binds with an affinity greater than M

[0135] A subject ABCG2-binding domain exhibits substantially no binding to epitopes formed by amino acids in other related but divergent proteins, such as related but divergent EPs. Binding of a subject ABCG2-binding domain to an epitope formed by amino acids in a related but divergent protein is generally nonspecific binding with substantially lower affinity than the specific binding of the ABCG2-binding domain to an epitope in ABCG2. 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.

[0136] A subject ABCG2 binding domain can reduce transport of a molecule via the ABCG2 transporter. For example, a subject ABCG2 binding domain can reduce transport by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the extent of transport in the absence of the ABCG2 binding domain.

[0137] A subject CD47 binding domain specifically binds to one or more epitopes of CD47. Thus, the epitopes are CD47 epitopes. The size of the CD47 epitope bound by an anti-CD47 binding domain can vary, including when the CD47 epitope is formed by a polypeptide having a contiguous stretch of the CD47 sequence, which can range from 4 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, 4-10 aa, 5 aa-10 aa, 6 aa-10 aa, 4 aa-8 aa, 5 aa-8 aa, 6 aa-8 aa, etc.

[0138] In some embodiments, the CD47 epitope is, for example, the human CD47 sequence: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE (SEQ ID NO: 46), or a rodent CD47 sequence, for example, mouse CD47 SEQ ID NO: MWPLAAALLLGSCCCGSAQLLFSNVNSIEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTTTDQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTVSWFSPNEKILIVIFPILAILLFWGKFGILTLKYKSSHTNKRIILLLVAGLVLTVIVVVGAILLIPGEKPVKNASGLGLIVISTGILILLQYNVFMTAFGMTSFTIAILITQVLGYVLALVGLCLCIMACEPVHGPLLISGLGIIALAELLGLVYMKFVASNQRTIQPPRNR (SEQ ID NO: 47), or a non-human primate sequence, for example, the Pongo abelii (Sumatran orangutan) sequence: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLIITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLNSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE (SEQ ID NO: 48), or the Macaca mulatta (rhesus monkey) sequence: MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTAPANFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELT REGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLMITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAI The CD47 sequence can be formed by a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of the CD47 sequence, including, but not limited to, LVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE (SEQ ID NO: 49).

[0139] A subject anti-CD47 binding domain exhibits high affinity binding to CD47. For example, a subject CD47 binding domain may bind to CD47 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 subject CD47 binding domain binds to an epitope present on CD47 with an affinity of approximately 10 M or greater. -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 binds with an affinity greater than M

[0140] A subject CD47-binding domain exhibits substantially no binding to epitopes formed by amino acids in other related but distinct sequences of proteins, such as related but distinct sequences of immune checkpoint markers. Binding of a subject CD47-binding domain to an epitope formed by amino acids in a related but distinct sequence of proteins is generally nonspecific binding with substantially lower affinity than the specific binding of the CD47-binding domain to an epitope in CD47. A substantially lower affinity is generally at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower affinity.

[0141] A subject CD47 binding domain can reduce binding of a CD47 binding partner to CD47, including, for example, thrombospondin-1 (TSP-1), signal-regulatory protein alpha (SIRPα), and integrins (e.g., integrin avb3). For example, a subject CD47 binding domain can reduce binding of a CD47 binding partner by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the degree of binding in the absence of the CD47 binding domain.

[0142] In some embodiments, the subject antibodies comprise a heavy chain FR1 region, an HCDR1, a heavy chain FR2 region, an HCDR2, a heavy chain FR3 region, an HCDR3, and a heavy chain FR4 region. In some of these embodiments, each of the FR regions is a mammalian FR region, including, for example, a human FR region. In some embodiments, the subject antibodies comprise a light chain FR1 region, an LCDR1, a light chain FR2 region, an LCDR2, a light chain FR3 region, an LCDR3, and a heavy chain FR4 region. In some of these embodiments, each of the FR regions is a mammalian FR region, including, for example, a human FR region.

[0143] In some embodiments, the subject antibodies comprise an anti-ABCG2 heavy chain sequence comprising charge-to-charge swap (KK) modifications to the Fc region, and an anti-CD47, anti-ErbB2, anti-EGFR, anti-PD-L1 heavy chain sequence comprising charge-to-charge swap (DD) modifications, particularly an anti-CD47 heavy chain sequence with charge-to-charge swap (DD) modifications, the sequence being: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 50). In some cases, the subject antibodies may comprise alternative heterodimeric Fc pairing strategies other than charge pair substitutions.

[0144] Charge-to-charge swap modification refers to a substitution in which one heavy chain is modified to contain K392D and K409D substitutions and the other heavy chain is modified to contain E356K and D399K substitutions. Charge-to-charge swapped chains favor heterodimer formation with each other. The numbering of amino acid substitutions follows the EU numbering system for Ig HC.

[0145] 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.

[0146] Linkers suitable for use in 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, arylacetylene, 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.

[0147] 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.

[0148] 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: 51), and GGGS n(SEQ ID NO: 52), 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: 53), GGSGG (SEQ ID NO: 54), GSGSG (SEQ ID NO: 55), GSGGG (SEQ ID NO: 56), GGGSG (SEQ ID NO: 57), GSSSG (SEQ ID NO: 58), 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.

[0149] In some embodiments, the subject antibody is "humanized." The term "humanized antibody" refers to an antibody that comprises variable region framework residues from a substantially human antibody chain (referred to as the acceptor immunoglobulin or antibody) and at least one CDR substantially from a non-human antibody (e.g., a rodent (e.g., a murine antibody), a non-human primate, etc.) (referred to as the donor immunoglobulin or antibody). See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761, WO 90 / 07861, and U.S. Pat. No. 5,225,539. The constant region, if present, can be substantially or entirely from a human immunoglobulin. In some embodiments, the subject antibody comprises one or more ABCG2 CDRs and one or more CD47 CDRs, and one or more FR regions from a human antibody. Methods for producing humanized antibodies are known in the art. See, e.g., U.S. Patent No. 7,256,273.

[0150] Substitution of mouse 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 mouse 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 mouse 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 those 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).

[0151] Now that the complementarity-determining regions of the murine donor immunoglobulin and the appropriate 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 murine for human amino acid residues should be minimized because 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 humanized antibodies 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.

[0152] 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-native juxtaposition of the murine CDR regions with the human variable framework regions can result in non-native conformational constraints, resulting in reduced binding affinity unless corrected by substitution of specific amino acid residues.

[0153] 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.

[0154] CDR and framework regions are as defined by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). An alternative structural definition has been proposed by Chothia et al., J. Mol. Biol. 196:901 (1987), Nature 342:878 (1989), and J. Mol. Biol. 186:651 (1989) (collectively referred to as "Chothia"). Amino acids present in murine antibodies can be selected for substitution into humanized antibodies if the framework residues defined by Kabat above constitute the structural loop residues defined by Chothia above. Residues "adjacent to the CDR region" include amino acid residues located immediately adjacent to one or more CDRs in the primary sequence of the humanized immunoglobulin chain, e.g., the CDRs defined by Kabat or Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids, in particular, may interact with amino acids in the CDRs and, if selected from the acceptor, distort the donor CDRs and reduce affinity. Moreover, adjacent amino acids may directly interact with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to maintain all of the antigen contacts that provide affinity in the original antibody.

[0155] 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 in tandem via a linker of about 2 amino acids to about 10 amino acids in length, e.g., a linker of 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length. Suitable linkers include, for example, (Gly) x (SEQ ID NO: 69), where x is an integer from 2 to 10. Other suitable linkers are discussed above. In some embodiments, each of the scFv monomers in a subject scFv multimer is humanized, as described above.

[0156] 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.

[0157] 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.

[0158] The antibody of interest can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the antibody of interest, a synthetic polymer, a carbohydrate, 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-di-(3',2'-pyridyldithio)propionamido]butane (DPDPB).

[0159] Compositions and Formulations The present disclosure provides compositions comprising a subject antibody. In addition to the subject antibody, the subject antibody compositions can include one or more of the following: a salt, such as NaCl, MgCl, KCl, MgSO, etc.; a buffer, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a surfactant, such as a non-ionic surfactant such as Tween-20; a protease inhibitor; glycerol, etc.

[0160] Compositions of the present disclosure also include pharmaceutical compositions comprising the multispecific 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 rate of cancer growth in a subject, or an improvement in cancer symptoms. Generally, the desired result is at least a reduction in cancer symptoms, a reduction in cancer growth, or a reduction in cancer size compared to a control. The subject antibody can be delivered or formulated in a manner that avoids the blood-brain barrier. In some cases, the antibody can include a delivery enhancer, where such an enhancer can facilitate crossing the blood-brain barrier, including, for example, increased permeability to enable efficient transdermal delivery. Useful delivery enhancers include, but are not limited to, for example, celeport, regadenoson, borneol, puerarin, propylene glycol, oleic acid, azone, N-methylpyrrolidone, Tween 80, limonene, lipid-based nanoparticles (NPs), liposomes, niosomes, transfersomes, ethosomes, dendrimers, micelle NPs, polymeric nanostructures, metallic nanostructures, magnetic nanostructures, recombinant human hyaluronidase, and the like.

[0161] 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.

[0162] 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.

[0163] For oral preparations, the subject antibodies can be used alone or in combination with suitable additives to form tablets, powders, granules, or capsules, for example, with conventional additives such as lactose, mannitol, corn starch, or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; with disintegrating agents such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, as needed, diluents, buffers, wetting agents, preservatives, and flavoring agents. In some cases, oral delivery of the antibody can be enhanced by complexing the antibody with a suitable hydrogel.

[0164] 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.

[0165] 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).

[0166] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and lyophilized preparations are reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equal to the volume removed during lyophilization), but solutions containing antimicrobial agents can be used to produce pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0167] 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.

[0168] Aqueous antibody formulations can be prepared in pH buffer solutions at pHs ranging from about 4.0 to about 7.0, 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.

[0169] An isotonicity agent may be included in the antibody formulation to adjust the tonicity of the formulation. Exemplary isotonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be preferred. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as physiological salt solution or serum. The isotonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 nM.

[0170] 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). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactants can range from about 0.001% to about 1% w / v.

[0171] 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.

[0172] 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).

[0173] 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.

[0174] As another example, a subject parenteral formulation is a liquid or lyophilized formulation comprising about 1 mg / mL to about 200 mg / mL of a subject antibody, 0.04% w / v Tween 20, 20 mM L-histidine, and 250 mM sucrose, and has a pH of 5.5.

[0175] As another example, a subject parenteral formulation may comprise: 1) 15 mg / mL of a subject antibody, 0.04% w / v Tween 20, 20 mM L-histidine, and 250 mM sucrose, and have a pH of 5.5; or 2) 75 mg / mL of a subject antibody, 0.04% w / v Tween 20, 20 mM L-histidine, and 250 mM sucrose, and have a pH of 5.5; or 3) 75 mg / mL of a subject antibody, 0.02% w / v Tween 20, 20 mM L-histidine, and 250 mM sucrose, and have a pH of 5.5. 2) 75 mg / mL of antibody of interest, 0.04% w / v Tween 20, 20 mM L-histidine, and 250 mM sucrose, and having a pH of 5.5; or 4) 75 mg / mL of antibody of interest, 0.04% w / v Tween 20, 20 mM L-histidine, and 250 mM trehalose, and having a pH of 5.5; or 6) 75 mg / mL of antibody of interest, 0.02% w / v Tween 20, 20 mM L-histidine, and 250 mM trehalose, and having a pH of 5.5.

[0176] As another example, a subject parenteral formulation may comprise: 1) 7.5 mg / mL of a subject antibody, 0.022% w / v Tween 20, 120 mM L-histidine, and 125 mM sucrose, and have a pH of 5.5; or 2) 37.5 mg / mL of a subject antibody, 0.02% w / v Tween 20, 10 mM L-histidine, and 125 mM sucrose, and have a pH of 5.5; or 3) 37.5 mg / mL of a subject antibody, 0.01% w / v Tween 20, 10 mM L-histidine, and 125 mM sucrose, and have a pH of 5.5; or 4) 37.5 mg / mL of a subject antibody, 0.02% w / v Tween 20, 10 mM L-histidine, and 125 mM sucrose, and have a pH of 5.5. 1) 37.5 mg / mL of antibody of interest, 0.01% w / v Tween 20, 10 mM L-histidine, and 125 mM trehalose, and having a pH of 5.5; or 5) 37.5 mg / mL of antibody of interest, 0.01% w / v Tween 20, 10 mM L-histidine, and 125 mM trehalose, and having a pH of 5.5; or 6) 5 mg / mL of antibody of interest, 0.02% w / v Tween 20, 20 mM L-histidine, and 250 mM trehalose, and having a pH of 5.5; or 7) 75 mg / mL of antibody of interest, 0.02% w / v Tween 20, 10 mM L-histidine, and 250 mM trehalose, and having a pH of 5.5. 7) 75 mg / mL of antibody of interest, 0.02% w / v Tween 20, 20 mM L-histidine, and 250 mM mannitol, and having a pH of 5.5; or 8) 75 mg / mL of antibody of interest, 0.02% w / v Tween 20, 20 mM L-histidine, and 140 mM sodium chloride, and having a pH of 5.5; or 9) 150 mg / mL of antibody of interest, 0.02% w / v Tween 20, 20 mM L-histidine, and 250 mM trehalose, and having a pH of 5.5; or 10) 150 mg / mL of antibody of interest, 0.02% w / v Tween 20, 20 mM L-histidine, and 250 mM trehalose, and having a pH of 5.5. 10) a liquid formulation comprising 10 mg / mL of antibody of interest, 0.01% w / v Tween 20, 20 mM L-histidine, and 40 mM sodium chloride, and a pH of 5.5.

[0177] 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.

[0178] Furthermore, the subject antibodies can be made into suppositories by mixing them with various bases, such as emulsifying bases or water-soluble bases. The subject antibodies can be administered rectally via a suppository. Suppositories can include vehicles such as cocoa butter, carbowax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0179] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, e.g., teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the subject antibody in a composition as a solution in sterile water, saline, or another pharmaceutically acceptable carrier.

[0180] 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.

[0181] Other modes of administration will also find use with the subject invention. For example, the subject antibodies can be formulated into suppositories, and in some cases, into aerosol and intranasal compositions. In suppositories, the vehicle composition will include traditional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w / w), for example, about 1% to about 2%.

[0182] Intranasal formulations typically contain a vehicle that does not cause irritation to the nasal mucosa or significantly interfere with ciliary function. Diluents such as water, aqueous saline, or other known substances can be used with the subject invention. Nasal formulations can also contain preservatives, such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant can be present to enhance absorption of the subject protein by the nasal mucosa.

[0183] The subject antibodies can be administered as injectable formulations. Typically, injectable compositions are prepared as liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. Preparations can also be emulsified, or the antibodies can be encapsulated in liposome vehicles.

[0184] 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. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the subject antibody appropriate to achieve the desired state in the treated subject.

[0185] 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.

[0186] In some embodiments, the subject antibody is formulated into a controlled-release formulation. Sustained-release preparations can be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Potential loss of biological activity and potential changes in immunogenicity of antibodies contained in sustained-release preparations can be prevented by using appropriate additives, controlling the water content, and developing specific polymer matrix compositions.

[0187] Controlled release within the scope of the present invention can be construed to mean any one of a number of extended-release dosage forms. The following terms: continuous release, controlled release, delayed release, depot, gradual release, extended release, programmed release, retarded release, proportional release, extended release, repository, inhibited delayed release, spaced release, sustained release, time-coat, timed release, delayed action, prolonged action, layered time action, prolonged action, prolonged action, repeated action, delayed action, sustained action, sustained-action drug, and extended release can be considered substantially equivalent to controlled release for purposes of the present invention. Further discussion of these terms can be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] A variety of hosts (the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") can be treated according to a subject method. Generally, such hosts 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 human.

[0197] Kits are provided having unit doses of the subject antibodies, e.g., oral or injectable doses. 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.

[0198] 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).

[0199] 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.

[0200] 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.

[0201] 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).

[0202] 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 viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655), adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997,Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997,Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999; Ali et al., Hum Mol Genet 5:591 594,1996, Srivastava in WO93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828, Mendelson et al., Virol. (1988) 166:154-165, and Flotte et al. al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al., J Virol 73:7812 7816, 1999), retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like.

[0203] As described above, a subject nucleic acid comprises a nucleotide sequence encoding a subject multispecific antibody. The subject nucleic acid can comprise nucleotide sequences encoding heavy- and light-chain CDRs. In some embodiments, the subject nucleic acid comprises nucleotide sequences encoding heavy- and / or light-chain CDRs, wherein the CDR-encoding sequences are interspersed with FR-encoding nucleotide sequences. In some embodiments, the subject nucleic acid comprises nucleotide sequences encoding heavy- and / or light-chain CD47 CDRs, wherein the CDR-encoding sequences are interspersed with FR-encoding nucleotide sequences. In some embodiments, the FR-encoding nucleotide sequences are human FR-encoding nucleotide sequences.

[0204] In some embodiments, a subject nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to an amino acid sequence provided herein.

[0205] 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.

[0206] 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, e.g., to a subject in need thereof. In some cases, the genetically modified cells may be used for the production, screening, and / or discovery of multispecific antibodies. Genetically modified cells may also, in some cases, include cells in which endogenous gene expression has been reduced, e.g., inhibited, knocked down, etc., or abrogated, e.g., knocked out. Genetically modified cells may also, in some cases, include cells in which gene expression has been enhanced, e.g., expression of an endogenous gene has been increased or expression of a heterologous gene has been increased.

[0207] 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.

[0208] Suitable mammalian cells include primary cells and immortalized cell lines, including human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), and HLHepG2 cells.

[0209] 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.

[0210] Suitable yeast cells or fungal or algal cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia species, Saccharomyces cerevisiae, Saccharomyces species, Hansenula polymorpha, Kluyveromyces species, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and the like.

[0211] Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus species, Salmonella species, Shigella species, and the like. See, e.g., Carrier et al. (1992) J. Immunol. 148:1176-1181, U.S. Patent No. 6,447,784, and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that can be used in the present invention include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, laboratory strains are non-pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus species, etc. In some embodiments, the host cell is Escherichia coli.

[0212] 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.

[0213] In some cases, useful cells expressing the multispecific antibodies of the present disclosure may include producer T cells. Non-limiting examples of producer T cells include those described in Tsai & Davila Oncoimmunology. (2016) 5(5):e1122158, the disclosure of which is incorporated herein by reference in its entirety. Producer T cells engineered to contain nucleic acid sequences encoding the multispecific antibodies of the present disclosure may, in some cases, be used to deliver the antibodies to a subject in need thereof.

[0214] Cells of the present disclosure also include cells that have been genetically modified to alter and / or modify the expression of one or more of ABCG2 and a TAA (e.g., CD47) in the cell. Such modified cells are useful for a variety of purposes, including assaying the binding of multispecific antibodies, and include, but are not limited to, those produced according to the description and methods provided herein. In some cases, ABCG2 may be knocked out or knocked down in a subject cell line. In some cases, a TAA (e.g., CD47) may be knocked out or knocked down in a subject cell line. In some cases, ABCG2 may be constitutively or inducibly overexpressed in a subject cell line. In some cases, a TAA (e.g., CD47) may be constitutively or inducibly overexpressed in a subject cell line. In some cases, both ABCG2 and a TAA (e.g., CD47) may be knocked down, knocked out, or constitutively or inducibly overexpressed in a subject cell line. Any convenient, suitable method for knocking down, knocking out, and / or overexpression may be used. The introduced nucleic acid can be stably integrated or can be transiently present.

[0215] In some embodiments, the cells of the present disclosure include genetically modified human cell lines that express a TAA (e.g., CD47) and contain an exogenous nucleic acid comprising a sequence encoding ABCG2 for overexpression of ABCG2. In such cells, TAA (e.g., CD47) expression can be endogenously or exogenously derived (i.e., introduced), and ABCG2 expression can be stable or transient. In some cases, the cell lines of the present disclosure that express a TAA (e.g., CD47) can be configured to generate genetically modified human cells that express a TAA (e.g., CD47) and stably overexpress ABCG2.

[0216] The cells and cell lines of the present disclosure can be cultured, for example, through the use of the culture methods described herein. In some cases, cells into which nucleic acids have been introduced to genetically modify the cells can be cultured to produce cell lines. Useful cell lines include, but are not limited to, genetically modified cell lines, including human cell lines, that express a TAA (e.g., CD47) and stably overexpress ABCG2.

[0217] The cells and cell lines of the present disclosure can be used in various methods of the present disclosure, e.g., as test samples, controls, etc. For example, in some cases, cells in which ABCG2 and / or a TAA (e.g., CD47) have been knocked out and / or knocked down can be used as reference cells to compare, e.g., binding of a multispecific antibody of the present disclosure. Other useful reference cells include, but are not limited to, non-cancerous cells, as well as normal cells and cells expressing normal levels of various proteins, including normal levels of ABCG2 and / or a TAA (e.g., CD47).

[0218] 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., multispecific antibodies, compositions and formulations, nucleic acids, expression vectors, cells, etc.

[0219] As summarized above, the methods of the present disclosure include contacting cancer cells with a multispecific antibody of the present disclosure to, for example, promote and / or enhance cancer cell killing. In some cases, cancer cell killing is mediated by an immune response or immune cells acting on the cancer cells as a result of opsonization of the cancer cells by bispecific targeting when two targets are co-expressed on the cancer cells. In some cases, cancer cell killing is mediated by an immune response or immune cells acting on the cancer cells as a result of, for example, masking or antagonizing a TAA (e.g., CD47) epitope present on the surface of the cancer cells by the multispecific antibody. In some cases, cancer cell killing is mediated by inhibition of cellular egress of the cancer cells as a result of, for example, ABCG2 antagonism on the cancer cells by the multispecific antibody. In some cases, the cells contacted with the multispecific antibody can be multidrug-resistant cancer cells. Methods involving contacting cancer cells with a multispecific 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, immunotherapy, radiation therapy, etc.

[0220] 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.

[0221] 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.

[0222] 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 a subject multispecific antibody, alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy). Administration of the multispecific antibodies of the present disclosure may be by any convenient, suitable delivery route.

[0223] Embodiments of the present disclosure include a bispecific antibody molecule according to the preceding section of this specification for use in a method of treating cancer in a subject, the method comprising administering the antibody to the subject. The method comprises administering the antibody in combination with at least one additional active agent, the at least one additional active agent comprising a chemotherapeutic agent, a multidrug resistance transporter inhibitor, an immunotherapeutic agent, or a combination thereof. In certain embodiments, the at least one additional active agent is a chemotherapeutic agent, optionally the chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate. Substrates for ABCG2 include topoisomerase II inhibitors (e.g., mitoxantrone, anthracyclines: doxorubicin, epirubicin, etc.), topoisomerase I inhibitors (e.g., camptothecin analogs: topotecan, gimatecan, etc.), tyrosine kinase inhibitors (e.g., gefitinib), etc.

[0224] Also disclosed are chemotherapeutic agents for use in methods of treating cancer in a subject, the methods comprising administering to the subject a chemotherapeutic agent in combination with an antibody described herein, optionally wherein the chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate.

[0225] Thus, administration includes, but is not limited to, for example, delivery of the antibody by injection, delivery of the antibody by infusion, delivery of a nucleic acid or expression vector encoding the multispecific antibody, delivery of the antibody by administering to a subject cells that express and secrete the multispecific antibody, etc. Administration of an agent, a nucleic acid encoding the agent, a cell expressing the agent, etc. can include contacting with the agent, contacting with the nucleic acid, contacting with the cell, etc.

[0226] In some embodiments, an effective amount of a subject multispecific 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 an adverse symptom 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 symptom in the absence of antibody treatment.

[0227] In some embodiments, an effective amount of a subject multispecific 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 multispecific antibody treatment.

[0228] In some cases, a subject may be treated systemically, involving the use of a subject multispecific antibody, with or without one or more additional reagents. "Systemic treatment," as used herein, means 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 at 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.

[0229] In some cases, a subject may be treated locally, involving the use of a subject multispecific 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 specifically directed 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, including tissue immediately adjacent to the tumor. Local treatments generally do not affect or target tissue 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 multispecific antibody include, but are not limited to, surgery, local radiation therapy, local cryotherapy, local laser therapy, local topical therapy, combinations thereof, etc.

[0230] In some embodiments, a subject treatment method involves administering a subject multispecific 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, for example, but are not limited to, radiation therapy, chemotherapy, immunotherapy, etc.

[0231] 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.

[0232] 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™), and the like. ™), oregovomab (OvaRex™), lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), atezolizumab (Tecentriq™), and conjugated antibodies, such as gemtuzumab ozogamicin (Mylortarg™), brentuximab vedotin (Adcetris™), 90Y-labeled Antibodies suitable for use in cancer therapy include, but are not limited to, ibritumomab tiuxetan (Zevalin™), 131I-labeled tositumomab (Bexxar™), and the like. Antibodies suitable for use in cancer therapy also include, but are not limited to, antibodies directed against tumor-associated antigens. Such antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate-binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Ley, VEGF, VEGFR, integrin alpha-V-beta-3, integrin alpha-5-beta-1, EGFR, ERBB2, ERBB3, PD-L1, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and the like.

[0233] 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 include, but are not limited to, venetoclax (Venclexta) which targets BCL2 (approved for use in chronic lymphocytic leukemia), vismodegib (Erivedge) which targets PTCH and smooth muscle (approved for use in basal cell carcinoma), vorinostat (Zolinza) which targets HDAC (approved for use in cutaneous T-cell lymphoma), and Ziv-aflibercept (Zaltrap) which targets PIGF and VEGFA / B (approved for use in colorectal cancer).

[0234] Suitable biological response modifiers 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.

[0235] Chemotherapeutic agents are non-peptide (i.e., non-proteinaceous) compounds that reduce the growth of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitroureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] "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).

[0244] 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 aforementioned Taxotere™ docetaxel) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, or paclitaxel-albumin).

[0245] 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 also included, including, but not limited to, those described in WO 98 / 58927, WO 98 / 13059, and U.S. Patent No. 5,824,701.

[0246] 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.

[0247] 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.

[0248] 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.).

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] In some cases, the methods of the present disclosure may include treating a subject having a cancer that is resistant to a first agent with an effective amount of a subject multispecific 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 may be resistant to a first chemotherapeutic agent, and the subject may be treated by administering an effective amount of a subject multispecific 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 may be used depending, for example, on the type of cancer being treated, the likelihood of developing resistance, etc.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] In some cases, the method can be used prophylactically for surveillance. For example, a subject in need thereof can be administered treatment with one or more of the 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.

[0259] 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 TAA (e.g., CD47, erbB2, or EGFR) above a predetermined threshold, or both.

[0260] In some cases, whether a subject is treated with a multispecific antibody of the present disclosure may depend on the results of TAA and / or ABCG2 testing. For example, in some cases, if the cancer expresses a TAA at or above a predetermined threshold, the subject may be treated with a multispecific antibody of the present disclosure; if the cancer expresses a TAA below the predetermined threshold, the subject may not be treated with a multispecific antibody, e.g., the subject may be treated with a conventional therapy for the relevant cancer without the subject multispecific antibody. For example, in some cases, if the cancer expresses ABCG2 at or above a predetermined threshold, the subject may be treated with a multispecific antibody; if the cancer expresses ABCG2 below the predetermined threshold, the subject may not be treated with a multispecific antibody, e.g., the subject may be treated with a conventional therapy for the relevant cancer without the subject multispecific antibody. In some cases, if the cancer expresses both a TAA and ABCG2 at or above a predetermined threshold, the subject may be treated with a multispecific antibody of the present disclosure; if the cancer expresses a TAA and ABCG2 below a predetermined threshold, the subject may not be treated with a multispecific antibody, e.g., the subject may be treated with a conventional therapy for the relevant cancer without the subject multispecific antibody.

[0261] Any convenient assay can be used to analyze ABCG2 and / or TAA 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. Useful predetermined thresholds for assessing expression of one or more markers and / or targets can be determined by any convenient and appropriate method, including comparing measured expression levels with a matched control. For example, in some cases, a useful predetermined threshold for the level of ABCG2 and / or TAA assayed in a sample can correspond to the level of ABCG2 and / or TAA measured in reference cells, such as healthy / normal cells. The TAA can be CD47, erbB2, EGFR, or PD-L1.

[0262] How to make it As summarized above, the methods of the present disclosure also include methods of making and / or identifying the multispecific antibodies described herein. The subject antibodies can be produced by any known method, such as conventional synthetic methods for protein synthesis, recombinant DNA methods, etc.

[0263] 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. Techniques for solid-phase synthesis are described by Barany and Merrifield, "Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A," Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., "Solid Phase Peptide Synthesis," 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10; and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, small, insoluble, porous beads are treated with functional units from which peptide chains are constructed. After repeated coupling / deprotection cycles, the free N-terminal amine of the bound solid phase is coupled to a single N-protected amino acid unit. This unit is then deprotected to expose a new N-terminal amine to which an additional amino acid can be coupled. The peptide remains immobilized on the solid phase and undergoes a filtration process before being cleaved.

[0264] 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.

[0265] Due to the degeneracy of the 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.

[0266] 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.

[0267] 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. For these prokaryotic hosts, one can also make expression vectors, typically containing expression control sequences (e.g., an origin of replication) compatible with the host cell. In addition, there are many different well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from phage lambda. The promoter typically controls expression, optionally with an operator sequence, and contains ribosome binding site sequences and the like, for initiating and completing transcription and translation.

[0268] 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.

[0269] 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, 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).

[0270] 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 cellular debris, macromolecules other than the subject antibody, etc.

[0271] In some embodiments, a method for generating a multispecific antibody of the present disclosure may include producing candidate antibodies and screening them for activity. Such a method may generate a multispecific antibody that specifically binds to cells expressing both ABCG2 and a TAA by using a series of steps. The steps of such a method may include producing a multispecific antibody or multiple antibodies, each of which contains or is expected to contain an ABCG2-binding domain and a TAA-binding domain; contacting a first test cell expressing ABCG2 and a TAA with the multispecific antibody or multiple antibodies; contacting a second test cell expressing either ABCG2 or a TAA with the multispecific antibody or multiple antibodies; comparing the binding of the multispecific antibody or multiple antibodies to the first cell with the 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 multiple antibodies as specific for cells expressing both ABCG2 and a TAA 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.

[0272] Various cells can be used in such methods, including, but not limited to, the cells described herein. In some cases, binding of the antibody to both cells expressing only ABCG2 and cells expressing only a TAA can be performed. For example, in some cases, the method may involve, in relation to the above steps, a second cell expressing ABCG2 but not a TAA, while the method may further include contacting a third cell expressing a TAA but not ABCG2 with the multispecific antibody.

[0273] 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 TAA antibody. Depending on the particular method used, various other or additional controls may be used as appropriate, as needed.

[0274] kit Aspects of the present disclosure also include kits. Kits may include, for example, any combination of multispecific antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. described herein. A subject kit may include one or more of a subject multispecific antibody, a nucleic acid encoding the same, or cells containing a subject multispecific nucleic acid. Kits may be configured for a variety of purposes, including, for example, therapeutic kits (e.g., the kit may include a 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0279] 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.

[0280] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996), Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 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).

[0281] Example 1: Generation of bispecific mAbs that sensitize cells to topotecan Materials and Methods Generation of stable ABCG2-overexpressing (Ox) cell lines To characterize both binding and in vitro efficacy, we developed a cell line stably overexpressing ABCG2. We utilized adherent 293T naive cells obtained from the American Type Culture Collection (ATCC). This cell line endogenously expresses ABCG2 at low to moderate levels on the cell surface, as characterized by flow cytometry using a commercially available ABCG2 antibody (R&D System, clone 5D3). 293T naive cells, 3T3 cells, or C6 cells (QZ) were transfected with ABCG2 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 ABCG2 cell surface expression. To ensure that untransfected cells would not grow in future cultures, bulk sorting of ABCG2-positive 293T cells was performed using fluorescence-activated cell sorting (FACS) on a FACSAriaI (BD Biosciences). Bulk-sorted 293T ABCG2-overexpressing cells were expanded and subsequently reconfirmed for ABCG2 overexpression.

[0282] 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.

[0283] 293 cells were used for transient production of bispecific mAbs. 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.

[0284] 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). For standard antibody expression, a ratio of 1:2 (heavy chain:light chain) was used.

[0285] Approximately 6 days after transfection, cells were harvested by centrifugation. Specifically, 1 μg of total encoding DNA per ml of transfected culture was diluted in Opti-MEM® medium (Life Technologies) and incubated with Expifectamine reagent (Life Technologies) in the same medium for 20 minutes. The mixture was then added to Expi293® cells growing in suspension in Expi293® Expression Medium (Life Technologies) at 37°C under 8% CO2 in air at 2.5 million cells / mL. After 6 days, the medium containing the antibody construct was harvested by centrifugation.

[0286] Construction of human-mouse sequences of tested molecules (human Fc, mouse Fv, 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, that 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. Different antibody chains were cloned into different vectors.

[0287] 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.

[0288] 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: TAA 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 can pair to provide acceptable binding for both Fab anti-TAA and Fab anti-ABCG2 (aABCG2), and its use allowed for avoiding mispairing of LCs. 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, the polypeptide chains or antibody halves directed against the target are assembled as bispecific antibodies through charge-pair substitutions in the CH3 domain: one heavy chain contains K392D and K409D substitutions ("DD"), and the other contains E356K and D399K substitutions ("KK").

[0289] Variable heavy and light chain fragments from mouse hybridoma sequences were available and cloned in the same background of leader sequences and constant regions.

[0290] 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) or KT9 (293T-KT9OX) 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 96-well microtiter plates 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.

[0291] 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 at 5000 cells / well in 0.05 mL of assay medium (DMEM + 10% FBS) in white, flat-bottom 96-well tissue culture plates. 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 fumitremordin C (FTC). An equal volume (0.05 mL) of the topotecan / antibody mixture was added to 293T_ABCG2_OX cells in the 96-well plate. The plate was then incubated at 37°C in 5% CO2. After approximately 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%.

[0292] Xenograft studies 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.

[0293] 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.

[0294] 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.

[0295] array The anti-ABCG2 5D3 antibody variable heavy (VH) chain sequence is as follows: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5)

[0296] Humanized versions of the 5D3 VH chain (v1 and v2) were generated.

[0297] The humanized 5D3 VHv1 sequence is as follows: EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWMGYINFDGGTTYNPSLRGRITISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 6)

[0298] The humanized 5D3 VHv2 sequence is as follows: EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWIGYINFDGGTTYNPSLRGRVTISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 7)

[0299] The anti-ABCG2 5D3 antibody variable light chain sequence is as follows: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1)

[0300] A humanized version of the 5D3 VL chain (v1) was generated.

[0301] The humanized 5D3 VLv1 sequence is as follows: DIQLTQSPSSLSASVGDRVTITCKASGYILNRLAWYQQKPGKAPKLLISGATSLETGFPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPWTFGGGTKLEIK (SEQ ID NO: 59)

[0302] The sequence encoding CD47: Homo sapiens CD47 molecule (CD47), transcript variant 1, mRNA NCBI reference sequence: NM_001777.3 is available.

[0303] The anti-CD47 5F9 antibody variable heavy chain sequence is as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 8)

[0304] The anti-CD47 5F9 antibody variable light chain sequence is as follows: DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADVGVYYCFQGSHVPYTFGQGTKLEIK (SEQ ID NO: 60)

[0305] The anti-erbB2 antibody, pertuzumab, heavy and light chain sequences are as follows: Pertuzumab heavy chain sequence: TIFF0007768902000002.tif53161 The VH chain is shown in bold and underlined.

[0306] Pertuzumab light chain sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 61)

[0307] The anti-erbB1 antibody, necitumumab, heavy and light chain sequences are as follows: Necitumumab heavy chain sequence: TIFF0007768902000003.tif49161 The VH chain is shown in bold and underlined.

[0308] Necitumumab light chain sequence: TIFF0007768902000004.tif30160 The VL chain is shown in bold and underlined.

[0309] The anti-PD-L1 antibody, atezolizumab, has the following variable heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 27)

[0310] result Figure 1 shows that 293T cells overexpressing ABCG2 have increased sensitivity to topotecan in the presence of the anti-ABCG2 antibody 5D3 and a bispecific antibody that binds to ABCG2 and CD47. The bispecific antibody 5D3DDKT14KK5D3 contains the heavy chain (HC) and light chain (LC) from the anti-ABCG2 antibody 5D3 and the HC from the anti-CD47 antibody 5F9. The bispecific antibody 5D3hVHv1DDKT14KK5D3hVLv1 contains the HC and LC from the anti-ABCG2 antibody 5D3 and the HC from the anti-CD47 antibody 5F9. The bispecific antibody 5D3hVHv2DDKT14KK5D3hVLv1 contains the HC and LC from the anti-ABCG2 antibody 5D3 and the HC from the anti-CD47 antibody 5F9. hVHv1 and hVHv2 refer to the humanized variable heavy chain types 1 and 2 of 5D3 HC, respectively. hVLv1 refers to the humanized variable light chain type 1 of 5D3 LC. KT14 refers to CD47. DD and KK refer to charge pair substitutions that enhance pairing between 5D3 HC and 5F9 HC. IC50 values ​​are in nM.

[0311] Figure 2 shows the binding of 5D3 and humanized 5D3 antibodies to 293T cells stably transfected to express human ABCG2 (293T-G2OX), along with the corresponding EC50 values. The results indicate that humanization does not significantly interfere with the efficacy of the 5D3 antibody.

[0312] Figure 3 shows the binding of the humanized ABCG2 / KT9 bispecific antibody to 3T3 cells stably transfected to express ABCG2 (3T3-G2), 293T cells stably transfected to express human ABCG2 (293T_ABCG2_OX), and 293T cells stably transfected to express human KT9 (293T-KT9OX). KT9 is the anti-PD-L1 antibody atezolizumab, and 5D3 is the anti-ABCG2 antibody. The humanized bispecific antibodies 5D3hVH-v1DD KT9KK 5D3hVL-v1 and 5D3hVH-v2DD KT9KK 5D3hVL-v1 contain the HC and LC from the anti-ABCG2 antibody 5D3 and the HC from the anti-PD-L1 antibody KT9. The results show that the humanized bispecific antibodies retain the ability to bind to their targets ABCG2 and PD-L1, respectively.

[0313] Figure 4 shows the binding of the humanized 5D3 / KT9 / 5D3 bispecific antibody to 293T cells stably transfected with ABCG2 (293T-G2OX), KT9 (293T-KT9OX), and both ABCG2 and KT9 (293T-G2KT9OX), along with the corresponding EC50 binding affinities. The results indicate that the humanized antibodies tested retain the ability to bind to their targets.

[0314] Figure 5 shows the results of a xenograft study in which the cytotoxic activity of the ABCG2 / PD-L1 bispecific antibody (5D3 / KT9) alone or in combination with topotecan was tested in the HT1376 (ATCC CRL-1472) human bladder epithelial carcinoma cell line. The bispecific antibody is shown to be effective both as a single agent and in combination with topotecan.

[0315] The present disclosure includes the following embodiments. Embodiment 1 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; wherein the VL chains each comprise an antigen-binding site for ABCG2, the first VH chain comprises an antigen-binding site for ABCG2, 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 VL chains; A bispecific antibody molecule, wherein the bispecific antibody binds to cancer cells expressing both ABCG2 and the TAA, but exhibits reduced binding to non-cancer cells expressing ABCG2 and / or the TAA. Embodiment 2 the antigen-binding sites of the two VL chains have the sequences: 2. The bispecific antibody molecule of embodiment 1, comprising light chain CDR1-3 (LCDR1-3) of the VL chain having: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1). Embodiment 3 3. The bispecific antibody molecule of embodiment 2, wherein the two VL chains comprise LCDR1-3, wherein LCDR1 comprises the sequence KASGYILNRLA (SEQ ID NO: 2), LCDR2 comprises the sequence GATSLET (SEQ ID NO: 3), and LCDR3 comprises the sequence QQYWSTPWT (SEQ ID NO: 4). Embodiment 4 the two VL chains having the sequences: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1), or 4. The bispecific antibody molecule of any one of embodiments 1 to 3, comprising an amino acid sequence having at least 90%, at least 95%, or at least 99% identity thereto: DIQLTQSPSSLSASVGDRVTITCKASGYILNRLAWYQQKPGKAPKLLISGATSLETGFPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPWTFGGGTKLEIK (SEQ ID NO: 59). Embodiment 5 the antigen-binding site of the first VH chain has the sequence: 10. The bispecific antibody molecule of any one of the preceding embodiments, comprising heavy chain CDR1-3 (HCDR1-3) of the VH chain having: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5). Embodiment 6 the antigen-binding site of the first VH chain comprises heavy chain CDRs 1 to 3 (HCDRs 1 to 3); (i) the HCDR1 comprises the sequence: SDYAWN (SEQ ID NO: 63); (ii) the HCDR2 comprises the sequence: YINFDGGTTYNPSLRG (SEQ ID NO: 64); (iii) The bispecific antibody molecule of any one of embodiments 1 to 4, wherein said HCDR3 comprises the sequence: FYGAKGTLDY (SEQ ID NO: 65). Embodiment 7 the first VH chain having the amino acid sequence: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5), EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWMGYINFDGGTTYNPSLRGRITISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 6), or 7. The bispecific antibody molecule of any one of embodiments 1 to 6, comprising: EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWIGYINFDGGTTYNPSLRGRVTISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 7), or an amino acid sequence with at least 90%, at least 95%, or at least 99% identity thereto. Embodiment 8 10. The bispecific antibody molecule of any one of the preceding embodiments, wherein said first and / or second VH chain is humanized and / or said VL chain is humanized. Embodiment 9 10. The bispecific antibody molecule of any one of the preceding embodiments, wherein the second VH chain is derived from a monospecific antibody molecule that binds to said TAA, and wherein the affinity of the bispecific antibody molecule for said TAA when paired with one of the light chains is at least two-fold less than the affinity of the monospecific antibody molecule for the TAA from which the VH chain is derived. Embodiment 10 10. The bispecific antibody molecule of any one of the preceding embodiments, wherein said TAA is CD47. Embodiment 11 the antigen-binding site of the second VH chain has the amino acid sequence: 11. The bispecific antibody molecule of embodiment 10, comprising HCDRs 1-3 of the VH chain comprising QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 8). Embodiment 12 12. The bispecific antibody molecule of any one of embodiments 1 to 11, wherein the second VH chain comprises an HCDR1 comprising the sequence: NYNMH (SEQ ID NO: 9), an HCDR2 comprising the sequence: TIYPGNDDTSYNQKFKD (SEQ ID NO: 10), and an HCDR3 comprising the sequence: GGYRAMDY (SEQ ID NO: 11). Embodiment 13 the second VH chain has the amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 8), EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 66), EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 67), or 13. The bispecific antibody molecule of any one of embodiments 1 to 12, comprising QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 68), or an amino acid sequence with at least 90%, at least 95%, or at least 99% identity thereto. Embodiment 14 10. The bispecific antibody molecule of any one of embodiments 1 to 9, wherein said TAA is the receptor tyrosine-protein kinase erbB-1. Embodiment 15 the antigen-binding site of the second VH chain has the amino acid sequence: 15. The bispecific antibody molecule of embodiment 14, comprising HCDRs 1-3 of the VH chain comprising QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 22). Embodiment 16 15. The bispecific antibody molecule of embodiment 14, wherein the antigen-binding site of the second VH chain comprises an HCDR1 comprising the sequence: SGDYYWS (SEQ ID NO: 19), an HCDR2 comprising the sequence: YIYYSGSTDYNPSLKS (SEQ ID NO: 20), and an HCDR3 comprising the sequence: VSIFGVGTFDY (SEQ ID NO: 21). Embodiment 17 the second VH chain has the amino acid sequence: 17. The bispecific antibody molecule of any one of embodiments 14 to 16, comprising QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 22), or an amino acid sequence with at least 90%, at least 95%, or at least 99% identity thereto. Embodiment 18 10. The bispecific antibody molecule of any one of embodiments 1 to 9, wherein said TAA is the receptor tyrosine-protein kinase erbB-2. Embodiment 19 the antigen-binding site of the second VH chain has the amino acid sequence: 19. The bispecific antibody molecule of embodiment 18, comprising HCDRs 1-3 of the VH chain comprising EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 12). Embodiment 20 19. The bispecific antibody molecule of embodiment 18, wherein the antigen-binding site of the second VH chain comprises an HCDR1 comprising the sequence: DYTMD (SEQ ID NO: 13), an HCDR2 comprising the sequence: DVNPNSGGSIYNQRFKG (SEQ ID NO: 14), and an HCDR3 comprising the sequence: NLGPSFYFDY (SEQ ID NO: 15). Embodiment 21 the second VH chain has the amino acid sequence: 21. The bispecific antibody molecule of any one of embodiments 18 to 20, comprising EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 12), or an amino acid sequence with at least 90%, at least 95%, or at least 99% identity thereto. Embodiment 22 10. The bispecific antibody molecule of any one of embodiments 1 to 9, wherein said TAA is Programmed Death-Ligand 1 (PD-L1). Embodiment 23 the antigen-binding site of the second VH chain has the amino acid sequence: 23. The bispecific antibody molecule of embodiment 22, comprising HCDRs 1-3 of the VH chain comprising EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 27). Embodiment 24 23. The bispecific antibody molecule of embodiment 22, wherein the antigen-binding site of the second VH chain comprises an HCDR1 comprising the sequence: DSWIH (SEQ ID NO: 28), an HCDR2 comprising the sequence: WISPYGGSTYYADSVKG (SEQ ID NO: 29), and an HCDR3 comprising the sequence: RHWPGGFDY (SEQ ID NO: 30). Embodiment 25 the second VH chain has the amino acid sequence: 25. The bispecific antibody molecule of any one of embodiments 22 to 24, comprising EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 27), or an amino acid sequence with at least 90%, at least 95%, or at least 99% identity thereto. Embodiment 26 10. The bispecific antibody molecule of any one of the preceding embodiments, wherein the antibody comprises an Fc domain that has been modified to reduce or abrogate binding of the antibody to one or more Fcγ receptors. Embodiment 27 10. The bispecific antibody molecule of any one of the preceding embodiments for use in a method of treating cancer in a subject, said method comprising administering said antibody to said subject. Embodiment 28 27. The bispecific antibody molecule for use according to embodiment 26, wherein said method comprises administering said antibody in combination with at least one additional active agent, wherein said 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 29 28. The bispecific antibody molecule for use according to embodiment 27, 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 30 A chemotherapeutic agent for use in a method of treating cancer in a subject, the method comprising administering to the subject the chemotherapeutic agent in combination with the antibody of any one of embodiments 1 to 26, optionally wherein the chemotherapeutic agent is taxol, a vinca alkaloid, or an anthracycline. Embodiment 31 30. The bispecific antibody molecule for use according to any one of embodiments 27 to 29, wherein said subject to be treated has a cancer which has been determined to be resistant to said chemotherapeutic agent. Embodiment 32 27. A method of treating a subject for cancer, comprising administering to the subject a therapeutically effective amount of a bispecific antibody molecule of any of embodiments 1 to 26. Embodiment 33 33. The method of embodiment 32, wherein the method comprises administering the bispecific 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 34 The method of embodiment 33, wherein the at least one additional active agent is a chemotherapeutic agent, optionally wherein the chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate. Embodiment 35 34. The method of embodiment 33, wherein the subject being treated has a cancer that has been determined to be resistant to treatment with the chemotherapeutic agent. Embodiment 36 1. A pharmaceutical composition comprising: An antibody according to any one of embodiments 1 to 26, and a pharmaceutically acceptable excipient. Embodiment 37 37. The pharmaceutical composition of embodiment 36, further comprising at least one additional active agent. Embodiment 38 38. The pharmaceutical composition of embodiment 37, wherein the at least one additional active agent comprises an immunotherapeutic agent. Embodiment 39 The pharmaceutical composition of embodiment 38, wherein the chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate. Embodiment 40 40. The pharmaceutical composition of any one of embodiments 37-39, wherein said at least one additional active agent comprises an inhibitor of a multidrug resistance transporter. Embodiment 41 The pharmaceutical composition of embodiment 40, wherein the at least one additional active agent comprises an immunotherapeutic agent. Embodiment 42 One or more nucleic acids comprising one or more sequences encoding the antibody of any of the preceding embodiments. Embodiment 43 43. The one or more nucleic acids of embodiment 42, wherein the one or more sequences are operably linked to a promoter. EMBODIMENT 44 44. One or more recombinant expression vectors comprising one or more nucleic acids of embodiment 42 or 43. Embodiment 45 A mammalian cell genetically modified with one or more recombinant expression vectors of embodiment 44. Embodiment 46 The cell of embodiment 45, wherein the cell is an immune cell. Embodiment 47 The antibody or a nucleic acid encoding the antibody according to any one of embodiments 1 to 26, and at least one additional active agent. Embodiment 48 48. The kit of embodiment 47, 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 49 A method of killing cancer cells, comprising contacting said cancer cells with the antibody of any of embodiments 1-26. Embodiment 50 50. The method of embodiment 49, comprising administering at least one additional active agent. Embodiment 51 51. The method of embodiment 50, wherein the at least one additional active agent comprises a chemotherapeutic agent. Embodiment 52 52. The method of embodiment 50 or 51, wherein said method increases said killing of said cancer cells by at least 5% compared to contact with said at least one additional active agent alone. Embodiment 53 53. The method of any one of embodiments 49 to 52, wherein the cancer cells are multidrug-resistant cancer cells. EMBODIMENT 54 A method of treating a subject for cancer, comprising administering to the subject an antibody of any of embodiments 1 to 26 or a pharmaceutical composition of any of embodiments 35 to 41. Embodiment 55 55. The method of embodiment 54, wherein the subject has been previously treated for the cancer. Embodiment 56 56. The method of embodiment 54 or 55, wherein the cancer is drug-resistant or multi-drug-resistant. Embodiment 57 57. The method of embodiment 56, wherein the cancer is resistant to a chemotherapeutic agent. Embodiment 58 57. The method of embodiment 56, wherein the cancer is resistant to an immunotherapy agent. Embodiment 59 The method of any one of embodiments 54-56, wherein the cancer is resistant to an inhibitor of a multidrug resistance transporter. Embodiment 60 60. The method of any one of embodiments 54-59, further comprising administering to said subject at least one additional active agent. Embodiment 61 61. The method of embodiment 60, wherein said at least one additional active agent comprises a chemotherapeutic agent. Embodiment 62 62. The method of embodiment 61, wherein said chemotherapeutic agent is taxol, a vinca alkaloid, an anthracycline, etoposide, mitoxantrone, or methotrexate. Embodiment 63 The method of any one of embodiments 60-62, wherein said at least one additional active agent comprises an inhibitor of a multidrug resistance transporter. EMBODIMENT 64 The method of any one of embodiments 60-62, wherein the at least one additional active agent comprises an immunotherapeutic agent. Embodiment 65 The method of any one of embodiments 60-62, wherein the method increases the effectiveness of the at least one additional active agent compared to treatment with the at least one additional active agent alone. Embodiment 66 66. The method of embodiment 65, wherein said increased efficacy comprises at least a 5% increase in cancer cell killing. Embodiment 67 67. The method of any one of embodiments 54-66, further comprising analyzing the cancer sample to determine whether the cancer expresses ABCG2 above a predetermined threshold, a tumor-associated antigen (TAA) above a predetermined threshold, or both, optionally wherein the TAA comprises CD47, erbB1, erbB2, or PD-L1. Embodiment 68 68. The method of embodiment 67, wherein said predetermined threshold corresponds to the level of ABCG2 and / or TAA expressed by a reference cell. Embodiment 69 69. The method of embodiment 68, wherein ABCG2 and / or TAA are knocked out or knocked down in said reference cells. Embodiment 70 70. The method of embodiment 68 or 69, wherein the reference cells are non-cancerous cells. Embodiment 71 69. The method of embodiment 68, wherein the non-cancerous cells express normal levels of ABCG2 and / or TAA. Embodiment 72 72. The method of any one of embodiments 54-71, wherein if the cancer expresses ABCG2 and a TAA at or above the predetermined threshold, the subject is administered a multispecific antibody, and if the cancer expresses ABCG2 or a TAA below the predetermined threshold, the subject is treated with a conventional therapy without administering the multispecific antibody. Embodiment 73 A bispecific antibody molecule that binds to ATP-binding cassette subfamily G member 2 (ABCG2) and a tumor-associated antigen (TAA), a first VH chain and a first VL chain, each of which comprises an antigen-binding site for ABCG2, wherein the first VH chain comprises HCDRs 1 to 3 of the amino acid sequence shown in SEQ ID NO: 5, and the first VL chain comprises LCDRs 1 to 3 of the amino acid sequence shown in SEQ ID NO: 1; the TAA is PD-L1, and the antibody molecule further comprises a second VH chain and a second VL chain, wherein the second VH chain comprises HCDRs 1-3 of the amino acid sequence set forth in SEQ ID NO: 27, and the second VL chain comprises LCDRs 1-3 of the VL chain of an anti-PD-L1 antibody, such as atezolizumab; or the TAA is CD47, and the antibody molecule further comprises a second VH chain and a second VL chain, wherein the second VH chain comprises HCDRs 1 to 3 of the amino acid sequence set forth in SEQ ID NO: 8, and the second VL chain comprises LCDRs 1 to 3 of the VL chain of an anti-CD47 antibody such as 5F9; or the TAA is HER2 (ErbB2), the antibody molecule further comprises a second VH chain and a second VL chain, the second VH chain comprising HCDRs 1 to 3 of the amino acid sequence set forth in SEQ ID NO: 16, and the second VL chain comprising LCDRs 1 to 3 of the VL chain of an anti-HER2 antibody such as pertuzumab or trastuzumab; or the TAA is HER1 (ErbB1), the antibody molecule further comprises a second VH chain and a second VL chain, the second VH chain comprises HCDRs 1 to 3 of the amino acid sequence set forth in SEQ ID NO: 17, and the second VL chain comprises LCDRs 1 to 3 of the VL chain of an anti-HER1 antibody such as necizumab; The bispecific antibody molecule binds to cancer cells expressing both ABCG2 and the TAA, but exhibits reduced binding to non-cancer cells expressing ABCG2 and / or the TAA. 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 thereto without departing from the spirit or scope of the appended claims.

[0316] Thus, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which 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 the concepts contributed by the inventors to furthering this 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.

[0317] 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 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; each of the VL chains comprising an antigen-binding site for ABCG2 having the following sequence: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1) an antigen-binding site comprising light chain complementarity-determining regions 1-3 (LCDR1-3) of a VL chain having the first VH chain comprising an antigen-binding site for ABCG2, the first VH chain comprising the following sequence: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5) an antigen-binding site comprising heavy chain complementarity-determining regions 1-3 (HCDR1-3) of the VH chain having the second VH chain is an antigen-binding site for the TAA, The following array: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 8) wherein the TAA is CD47; or The following array: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 27) wherein the TAA is programmed cell death ligand 1 (PD-L1), the second VH chain binds to the TAA when paired with one of the VL chains; the bispecific antibody binds to cancer cells expressing both ABCG2 and the TAA, but exhibits reduced binding to non-cancer cells expressing ABCG2 or the TAA; wherein the CDRs are those defined by Kabat, Chothia, or MacCallum. Bispecific antibody molecule. b) the two VL chains comprise an LCDR1 comprising the sequence KASGYILNRLA (SEQ ID NO: 2), an LCDR2 comprising the sequence GATSLET (SEQ ID NO: 3), and an LCDR3 comprising the sequence QQYWSTPWT (SEQ ID NO: 4); and / or c) the two VL chains have the sequences: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLAWYQQKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLSISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIR (SEQ ID NO: 1), or DIQLTQSPSSLSASVGDRVTITCKASGYILNRLAWYQQKPGKAPKLLISGATSLETGFPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPWTFGGGTKLEIK (SEQ ID NO: 59), or comprises an amino acid sequence that has at least 90%, at least 95%, or at least 99% identity to said sequence; and / or e) the antigen-binding site of the first VH chain is (i) an HCDR1 comprising the sequence: SDYAWN (SEQ ID NO: 63); and (ii) an HCDR2 comprising the sequence: YINFDGGTTYNPSLRG (SEQ ID NO: 64); and (iii) an HCDR3 comprising the sequence: FYGAKGTLDY (SEQ ID NO: 65); and / or f) the first VH chain has the amino acid sequence: QVQLQESGPGLVKPSQSLSLTCTVTGFSITSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRISITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYWGQGTSVTVSS (SEQ ID NO: 5), EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWMGYINFDGGTTYNPSLRGRITISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 6), or EVQLQESGPGLVKPSETLSLTCTVSGFSITSDYAWNWIRQPPGKGLEWIGYINFDGGTTYNPSLRGRVTISRDTSKNQFSLKLSSVTAADTAVYYCATFYGAKGTLDYWGQGTLVTVSS (SEQ ID NO: 7), or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to said amino acid sequence, where: the second VH chain is derived from a monospecific antibody molecule that binds to the TAA, and the affinity of the bispecific antibody molecule for the TAA when paired with one of the light chains is at least two-fold less than the affinity of the monospecific antibody molecule for the TAA from which the VH chain is derived. The bispecific antibody molecule of claim 1.

3. The bispecific antibody molecule of claim 1 or 2, wherein the TAA is CD47. b) the second VH chain comprises an HCDR1 comprising the sequence NYNMH (SEQ ID NO: 9), an HCDR2 comprising the sequence TIYPGNDDTSYNQKFKD (SEQ ID NO: 10), and an HCDR3 comprising the sequence GGYRAMDY (SEQ ID NO: 11); and / or c) the second VH chain has the amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 8), EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYNMHWVRQAPGKGLEWMGTIYPGNDDTSYNQKFKDRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYRAMDYWGQGTLVTVSS (SEQ ID NO: 66), EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYNMHWVRQMPGKGLEWMGTIYPGNDDTSYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 67), or QVQLVQSGSELKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWMGTIYPGNDDTSYNQKFKDRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGGYRAMDYWGQGTTVTVSS (SEQ ID NO: 68), or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence. The bispecific antibody molecule of claim 3.

5. the TAA is programmed cell death ligand 1 (PD-L1); b) the antigen-binding site of the second VH chain comprises an HCDR1 comprising the sequence: DSWIH (SEQ ID NO: 28), an HCDR2 comprising the sequence: WISPYGGSTYYADSVKG (SEQ ID NO: 29), and an HCDR3 comprising the sequence: RHWPGGFDY (SEQ ID NO: 30); and / or c) the second VH chain has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 27), or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity thereto; A bispecific antibody molecule according to claim 1 or 2.

6. A bispecific antibody molecule according to any one of claims 1 to 5, wherein the antibody comprises an Fc domain that has been modified to reduce or prevent binding of the antibody to one or more Fcγ receptors.

7. A pharmaceutical composition comprising the bispecific antibody molecule of any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. 8. The pharmaceutical composition of claim 7 for use in a method of treating cancer or killing cancer cells in a subject, the method comprising administering to the subject or contacting cancer cells with a therapeutically effective amount of the pharmaceutical composition.

9. The pharmaceutical composition of claim 8, wherein the method comprises administering the bispecific antibody molecule 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.

10. The immunotherapeutic agent targets an immune checkpoint marker, and / or The chemotherapeutic agent i) an alkylating agent, a nitrosourea, an antimetabolite, an antitumor antibiotic, a plant alkaloid, or a steroid hormone; ii) is a topoisomerase inhibitor; The pharmaceutical composition of claim 9.

11. 11. The pharmaceutical composition of any one of claims 7 to 10, wherein the pharmaceutical composition further comprises 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, or an immunotherapeutic agent.

12. The immunotherapeutic agent targets an immune checkpoint marker, and / or The chemotherapeutic agent i) an alkylating agent, a nitrosourea, an antimetabolite, an antitumor antibiotic, a plant alkaloid, or a steroid hormone; ii) is a topoisomerase inhibitor; The pharmaceutical composition of claim 11.

13. The pharmaceutical composition of any one of claims 9 to 12, wherein the chemotherapeutic agent is a taxane, a vinca alkaloid, or an anthracycline.

14. One or more nucleic acids comprising one or more sequences encoding the bispecific antibody molecule of any one of claims 1 to 6.

15. 15. One or more recombinant expression vectors comprising one or more nucleic acids of claim 14.

16. 16. A mammalian cell genetically modified with one or more recombinant expression vectors of claim 15.

17. A bispecific antibody molecule according to any one of claims 1 to 6 or a nucleic acid according to claim 14, and at least one additional active agent.

18. 18. The kit of claim 17, 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.

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