Anti-ABCG2 antibodies and their use

Abstract

To provide an antibody molecule that specifically binds to ATP-binding cassette subfamily G member 2 (ABCG2). [Solution] Antibodies targeting the cell efflux pump ABCG2 are provided. Also provided are pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies. Methods of using antibodies to detect the presence or absence of ABCG2 expression, the level of ABCG2 expression, and / or to inhibit ABCG2 function in cells, e.g., tumor cells, are also disclosed. In addition, multispecific antibodies that bind to ABCG2 and polypeptides expressed on the surface of cancer cells are provided. The polypeptides may be the efflux pump MDR1 or cancer-related antigens. Also provided are methods for treating a subject for cancer, comprising administering to the subject an anti-ABCG2 antibody disclosed herein, or a multispecific antibody targeting both ABCG2 and cancer-related antigens or MDR1.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is U.S. Provisional Patent Application No. 63 / 034,806, filed on June 4, 2020. Claiming the benefit of priority, the application is incorporated herein by reference in its entirety.

[0002] Inclusion by referencing sequence listings provided as text files The sequence listing was created on May 24, 2021, and is a text file with a size of 266KB. The file "KNJY-004WO SEQ LIST_ST25.txt" is used in this specification. The contents of the text files are provided to the user. The entire contents of these text files are incorporated herein by reference. Born. [Background technology]

[0003] Drug resistance is a well-known phenomenon that occurs when a disease becomes resistant to drug treatment, and tumors This is a major and increasingly growing challenge in various fields of medicine, including ulcerology. Ip cancer is initially sensitive to chemotherapy, but over time, it becomes more difficult to treat. Furthermore, metabolic changes that promote DNA mutations and the inhibition, degradation, and enhanced elimination of drugs Resistance can be developed through other mechanisms, including those mentioned above.

[0004] The efflux pump (EP) is a protein expressed by living cells that effluxes various compounds. They have evolved to be naturally expelled from cells. ATP-binding cassette (ABC) transdextrin Members of the porter family of proteins are examples of EPs that enable drug efflux. The structure of transporters differs from protein to protein (for example, there are 49 known types in humans). There are BC family members), all of which are classified by the presence of two different domains - a highly conserved nucleotide-binding domain and a more variable transmembrane domain . The multidrug resistance protein 1 (MDR1) encoded by the ATP-binding cassette subfamily B member 1 (ABCB1) gene is 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 enables tumors to develop resistance to chemotherapeutic agents. Such resistance is often associated with enhanced efflux of chemotherapeutic agents from drug-resistant cells. This chemotherapeutic resistance is called multidrug resistance (MDR) when applied to two or more chemotherapeutic agents .

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

Summary of the Invention

[0007] Antibodies targeting the cell efflux pump ABCG2 are provided. Also provided are pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits containing or encoding such antibodies . Methods of using the antibodies to detect the presence or level of ABCG2 expression and / or inhibit ABCG2 function in cells, such as tumor cells, are also disclosed . In addition, multispecific antibodies that bind to cancer cells overexpressing ABCG2 and tumor-associated antigens (TAAs: tumor-associated antigen) on the cell surface are provided. In addition, ABCG tumor-associated antigen) on the cell surface are provided. In addition, ABCG A multispecific antibody is provided that binds to cancer cells that overexpress 2 and the efflux pump ABCB1. Furthermore, the anti-ABCG2 antibody or multispecific antibody disclosed herein is administered to the target. Methods for treating cancer are provided, including the following. [Brief explanation of the drawing]

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

[0009] definition The terms "antibody" and "immunoglobulin" refer to any isotype of antibody or immunoglobulin. Robulins are fragments of antibodies that retain specific binding to antigens, such as Fab, Fv, scFv, and Fd. Fragments including but not limited to Fab', Fv, and F(ab')2, chimeric antibodies, tethered antibodies, monoclonal antibodies, and antibodies containing only the heavy chain (e.g., VHH camelid antibodies) A fusion containing single-chain antibodies, bispecific antibodies, and the antigen-binding portion of an antibody and a non-antibody protein. It contains synthetic proteins. Antibodies, for example, are radioactive isotopes, enzymes that produce detectable products, Antibodies can be labeled with fluorescent proteins or other means to make them detectable. Antibodies are members of a specific binding pair. For example, in other parts such as biotin (a member of the biotin-avidin specific binding pair) It can be conjugated to. Antibodies also include polystyrene plates or beads, etc. Antibodies can be bound to solid supports, but are not limited to these. Antibodies may be monovalent or bivalent. Antibodies can be conjugated to the toxic portions of chemotherapeutic agents and other substances.

[0010] "Antibody fragment" is a part of an intact antibody, for example, the antigen-binding region of an intact antibody or Includes variable regions. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. Diabody; Linear antibody (Zapata et al., Protein Eng. 8) (10): 1057-1062 (1995)), antibodies containing only the heavy chain (e.g., VHH It contains single-chain antibody molecules (including camel antibodies) and multispecific antibodies formed from antibody fragments. The papain digestion of antibodies is called a "Fab" fragment, each having a single antigen-binding site. Two identical antigen-binding fragments, and the ability to easily crystallize. The remaining "Fc" fragment, which reflects the naming, is produced. Pepsin treatment reveals two antigen-binding sites. This results in an F(ab')2 fragment that possesses and can still crosslink the antigen.

[0011] "Fv" is the smallest antibody fragment containing complete antigen-recognition and antigen-binding sites. The region consists of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. It consists of a dimer. In this three-dimensional structure, the three CDRs of each variable domain interact, V The antigen-binding site is defined on the surface of the H-VL dimer. In summary, the six CDRs are used in antibodies. It confers antigen-binding specificity. However, a single variable domain (or specific to the antigen) Even half of the Fv, which contains only three typical CDRs, has the ability to recognize and bind to the antigen. This has a lower affinity than the entire binding site, which includes the three CDRs in each variable domain.

[0012] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment contains one or more cysteines from the antibody hinge region, and the heavy chain CH1 The addition of several residues at the carboxyl terminus of the domain results in the Fab' fragment being different from the others. They are different. Fab'-SH has a cysteine ​​residue in its constant domain that has a free thiol group. This is the nomenclature used herein for ab'. The F(ab')2 antibody fragment was originally inter It was generated as a paired Fab' fragment with a hinge cysteine. Other chemical formulations of the antibody fragment Scientific coupling is also known.

[0013] The "light chains" of antibodies (immunoglobulins) from any vertebrate species are their constant domains. Based on their amino acid sequences, two distinctly different types of cysts are called kappa and lambda. It can be assigned to one of them, depending on the amino acid sequence of the constant domain of those heavy chains. Therefore, immunoglobulins can be assigned to different classes. Five types of immunoglobulins The main classes are IgA, IgD, IgE, IgG, and IgM, and among these... Furthermore, there are subclasses (isotypes), for example, IgG1, IgG2, IgG3, It can be divided into IgG4, IgA, and IgA2.

[0014] "Single-chain Fv", "sFv", or "scFv" antibody fragments are antibodies of the V type. H and V L Domaine These domains are present in a single polypeptide chain. Several embodiments In this context, the Fv polypeptide is V H Domain and V L Polypeptide between the domain It further contains a ker, allowing sFv to form the desired structure for antigen binding. An overview of Fv, The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315. See Pluckthun in (1994).

[0015] The term "diabody" refers to a small antibody fragment that has two antigen-binding sites. This fragment has a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same port It is contained in the lipeptide chain (VH-VL). It enables pairing between two domains on the same chain. By using a linker that is too short, the domain becomes a complementary domain of another chain. It is paired with and forms two antigen-binding sites. The diabody is, for example, EP404. 097, WO93 / 11161, and Hollinger et al., Proc.N Completed by atl.Acad.Sci.USA,90:6444-6448(1993) It is described throughout.

[0016] As used herein, the term "affinity" refers to the reversible binding of two drugs. This refers to the equilibrium constant and is expressed as the dissociation constant (Kd). Affinity is the unrelated amino acid combination. At least 1 times greater than the affinity of the antibody against the column, at least 2 times greater, less Both are three times larger, at least four times larger, at least five times larger, and at least six times larger. , at least 7 times larger, at least 8 times larger, at least 9 times larger, at least 1 0 times larger, at least 20 times larger, at least 30 times larger, at least 40 times larger i, at least 50 times larger, at least 60 times larger, at least 70 times larger, less At least 80 times larger, at least 90 times larger, at least 100 times larger, or less Both can be 1000 times larger or more. Antibodies against target proteins The affinity is, for example, from about 100 nanomoles (nM) to about 0.1 nM, and from about 100 nM to about 1 pi It can be comolar (pM) or approximately 100 nM to approximately 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the 2-fold dissociation after dilution. This refers to resistance to a complex of more than one drug. It is characterized by "immunoreactivity" and "preferential binding." The terms used herein are interchangeable with respect to antibodies and / or antigen-binding fragments.

[0017] The term "bonding" includes covalent interactions, electrostatic interactions, hydrophobic interactions, and, for example, For example, interactions such as salt bridges and water bridges, and ionic and / or hydrogen bond interactions. This refers to the direct association between two molecules. ABCG2-specific antibodies are ABCG2 polypeptides. It specifically binds to epitopes within the molecule. Epitopes are formed by the continuous extension of amino acids. Linear epitopes formed, or non-linear epitopes formed by discontinuous elongation of amino acids. It can be a top or a structural epitope. Nonspecific binding occurs at approximately 10 -7 Parents under M Bonding by harmony, for example, 10 -6 M, 10 -5 M, 10 -4 Binding based on affinity, such as M. It refers to.

[0018] As used herein, the terms “CDR” or “complementarity-determining region” refer to heavy chains and This refers to discontinuous antigen-binding sites found within the variable regions of both the light chain polypeptide and the tertiary chain polypeptide. This is intended. CDR is a highly variable region and is called the "framework region (FR)". These are scattered along with more preserved regions. CDRs are by Kabat et al. J. Biol. Chem. 252:6609-6616 (1977), Kabat et al. al.,USDept.of Health and Human Service es,“Sequences of proteins of immunology al interest” (1991), Chothia et al., J. Mol. Biol.196:901-917(1987), and MacCallum et al. This is described by .,J.Mol.Biol.262:732-745(1996) Furthermore, their definitions include the presence or absence of duplicate amino acid residues when compared to one another. A subset of these is included. Nevertheless, the CDR of antibodies or their graft antibodies The application of any definition referring to a body or variant is limited to the definitions and uses herein. It is intended to be within the scope of the word. CD as defined by each of the references cited above. For comparison, the amino acid residues containing R are shown in Table 1 below. [Table 1]

[0019] As used herein, the term "framework" has the same meaning as the antibody variable region. When used, it refers to all amino acid residues outside the CDR region in the variable region of the antibody. The intention is that the variable domain framework generally consists of approximately 100-120 elements of length. Although the amino acid sequence is discontinuous, only these amino acids outside the CDR are mentioned. It is intended to be. When used herein, the term "framework area" is used. This is intended to mean each domain of the framework that is separated by the CDR. The VH chains are in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 can contain three CDRs and four FRs arranged from the N-terminus to the C-terminus. Similarly, the VL chain is in the following order: FR1, CDR1, FR2, CDR2, FR3, C DR3, FR4, and 3 CDRs and 4 FRs are arranged from the N-terminus to the C-terminus. It is possible.

[0020] As used herein, the term antibody refers to a tetramer consisting of two heavy chains and two light chains. The heavy and light chains are interconnected, for example, by disulfide bonds. (Heavy chain stationary) The region consists of three domains: CH1, CH2, and CH3. The light chain constant region is, It consists of a single domain called CL. The variable regions of the heavy and light chains interact with the antigen. Includes the synergistic region. The constant region of an antibody is typically the primary region of various cells in the immune system and complement system. It mediates the binding of antibodies to host tissues and factors that contain the components. The term "antibody" is The types of immunoglobulins are IgA, IgG, IgE, IgD, IgM, and their Includes subtypes. In some embodiments, the target antibody is an IgG isotype, e.g. For example, IgG1.

[0021] As used herein, the term "immunoglobulin" refers to immunoglobulin genes. Refers to a protein containing one or more polypeptides substantially encoded by [the specified entity]. The human immunoglobulin genes identified include kappa, lambda, alpha (IgA1 and IgA2) ), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mi This includes constant region genes and numerous immunoglobulin variable region genes. The globulin light chain (approximately 25kD or 214 amino acids) has a variable region gene at its N-terminus (approximately Encoded by 110 amino acids and a kappa or lambda constant region at the C-terminus. Long immunoglobulin heavy chains (approximately 50 kD or 446 amino acids) are inherited from the variable region at the N-terminus. The offspring (approximately 116 amino acids) and one of the other constant region genes mentioned above at the C-terminus, for example Then, it is coded by gamma (which codes for approximately 330 amino acids). Some actual In the application form, the target antibody contains full-length immunoglobulin heavy chains and full-length immunoglobulin light chains. nothing.

[0022] The term "antigen-binding fragment" refers to a type of full-length antibody that can specifically bind to an antigen. This refers to the above fragments. Examples of combined fragments include (i) Fab fragments (VL, VH, CL, and CH (ii) F(ab')² fragments (including, for example, monovalent fragments consisting of, 1 domain), A divalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region. (iii) Fd fragment (containing, for example, the VH and CH1 domains) (iv) Fv fragment (e.g., containing the VH and VL domains of a single arm of the antibody, e.g. (consisting of these), (v)dAb fragment (containing the VH domain, e.g., consisting of the same) (vi) Isolated CDRs, (vii) Single-strand Fv (scFv) (e.g., VH and VL) Domain pairs are linked by a synthetic linker using recombinant means to form a monovalent molecule. The antibody includes, for example, the VH and VL domains of a single arm, (consisting of these), ( viii) Diabody (VH and VL domains so that they do not form a monovalent molecule) For example, it consists of two scFvs that do not form a pair, and each of the scFvs is 1 (One VH pairs with the VL domain of another scFv to form a divalent molecule.)

[0023] The term "chimeric" antibody refers to a part of the heavy and / or light chain that is derived from a specific source or species. The antibodies originate from one source, while the heavy chain and / or light chain residues originate from a different source or species. It refers to.

[0024] "Human antibodies" are produced by humans or human cells, or are part of the human antibody repertoire. This corresponds to antibodies derived from non-human sources that utilize Lee or other human antibody coding sequences. It is a substance that has an amino acid sequence. This definition of a human antibody includes non-human antigen-binding residues. Clearly exclude t-induced antibodies.

[0025] The "Human Consensus Framework" is based on human immunoglobulin variable light chains (VLs) or other variable light chains. In selecting the most commonly present amino acid residues in the heavy chain (VH) framework sequence This is a framework (FR) that represents the selection of human immunoglobulin VL or VH sequences. The selection is from subgroups of variable domain sequences. Generally, subgroups of sequences are K abat et al., Sequences of Proteins of Imm unological interest,Fifth Edition,NIH Pu Blication 91-3242, Bethesda Md. (1991), vol. These are subgroups as shown in s.1-3. In one embodiment, with respect to VL, The group is Kappa I, as described by Kabat et al. above. Regarding morphology, for VH, the subgroup is Kabat et al., as mentioned above. This is subgroup III.

[0026] "Humanized" antibodies are derived from amino acid residues and human frameworks (FRs) from non-human CDRs. This refers to a chimeric antibody containing amino acid residues from [source]. At least a portion of the constant region of the humanized antibody is [value]. Derived from human antibodies, for example, human IgG1 antibodies. In a preferred embodiment, as specified herein. The antibody molecules disclosed herein include a variable heavy chain region and UniProt:P Human IgG1 stationary having the amino acid sequence shown in 01857-1, version 1. It includes a heavy chain containing a region. In a preferred embodiment, the antibody molecule disclosed herein is The light chain includes a variable light chain region and a human light chain constant region provided herein. In the embodiment, the human light chain constant region is UniProtKB / Swiss-Prot: This is the constant region of the human kappa light chain containing the amino acids shown in P01834.2. In this case, the human IgG1 heavy chain constant region present in the target antibody is mutated, for example, Fc function This may include substitutions that modulate the function of the LALAPG effector (L234A, By introducing the L235A and P329G) or N297A mutations, antibody-dependent cytotoxicity ( ADCC) may be reduced. The replacement numbering is based on the EU numbering system. The "numbering system" or "EU index" assigns residues within the constant region of the immunoglobulin heavy chain to It is commonly used when making references (for example, Kabat et al., Sequen ces of Proteins of Immunological Interes t,5th Ed.Public Health Service,National Institutes of Health, Bethesda, MD. (1991) (Reported EU index). "EU index like in Kabat" This refers to the numbering of residues in human IgG 1 EU antibodies.

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

[0028] The term "epitope" refers to a location on an immune system, such as an antibody, B cell, or T cell. Therefore, it refers to the region of the antigen that is recognized. For example, an epitope is the specific antigen to which an antibody binds. It is a specific domain.

[0029] "Isolated" antibodies are identified, separated, and / or recovered from components of their natural environment. It is an antibody. The contamination components in its natural environment are used in the diagnosis or treatment of the antibody. Substances that may interfere with the intended use include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. It may contain a sex solute. In some embodiments, the antibody is determined by (1) the Lowry method. When determined, the antibody weight is greater than 90%, greater than 95%, or greater than 98%, for example, 9 Up to 9%, (2) by using a spinning cup sequencer, N-terminus or internal (3) Coomassieble - Alternatively, using silver staining, sodium dodecyl sulfate - under reducing or non-reducing conditions. The material is purified to homogeneity using polyacrylamide gel electrophoresis (SDS-PAGE). The isolated antibody contains the antibody in insights within recombinant cells, which is the natural state of the antibody. This is because at least one component of the natural environment is absent. In some cases, isolation The antibodies are prepared by at least one purification step.

[0030] As used herein, the term "cytotoxic agent" means an agent that inhibits or inhibits cellular function. This refers to a substance that prevents and / or causes cell death or cell destruction. It is also called an "antitumor agent." The term "chemotherapeutic agent" refers to a drug used to treat cancer or other diseases or disorders. It can be a cytotoxic agent.

[0031] As used herein, terms such as “treatment” and “to treat” mean “to treat the desired drug.” This refers to obtaining physical and / or physiological effects. The effect is to completely eliminate or alleviate the disease or its symptoms. It can be preventive in the sense that it partially prevents the disease and / or the disease It may be therapeutic in that it partially or completely cures the resulting side effects. When used in writing, "treatment" encompasses any treatment of disease in mammals, including humans. (a) In subjects who are susceptible to disease but have not been diagnosed with it, the disease (b) to prevent the occurrence of the disease, i.e., to inhibit its onset. (c) to alleviate the disease, that is, to cause disease regression. include.

[0032] The terms "individual," "subject," "host," and "patient" as used interchangeably in this specification are... The terminology includes rodents (rats, mice), non-human primates, humans, dogs, cats, and ungulates (for example). This refers to mammals, including but not limited to horses, cows, sheep, pigs, and goats. .

[0033] "Therapeutic dose" or "effective dose" is the amount administered to a mammal or other subject to treat a disease. When this occurs, the amount of target-specific antibodies is sufficient to influence the treatment of the disease. This refers to the "therapeutic dose," which is determined by the antibody, the disease and its severity, and the age and body size of the person being treated. It changes depending on the weight, etc.

[0034] As used herein, the term "refractory" refers to a disease or condition that does not respond to treatment. With regard to cancer, "refractory cancer," as used herein, refers to cancer that does not respond to treatment. This refers to cancers that may be resistant at the start of treatment or that may become resistant during treatment. Refractory cancers can also be called drug-resistant cancers.

[0035] "Biological samples" encompass various sample types obtained from individuals and are used for diagnosis or monitoring. It can be used in assays. Definition: Blood and other fluid samples of biological origin, biopsy specimens. Solid tissue samples such as, or tissue cultures or cells derived therefrom and their offspring, are enclosed. Includes. The definition also includes any method after their acquisition, e.g., treatment with reagents, solubilization, Alternatively, the sample may include one that has been manipulated by enriching it with specific components such as polynucleotides. The term "biological sample" encompasses clinical samples, including cultured cells, cell supernatants, cell lysates, etc. This includes serum, plasma, biological fluids, and tissue samples.

[0036] The identity percentage between pairs of sequences is calculated by multiplying the number of matches in the pair by 100, including any gaps. It can be calculated by dividing by the length of the aligned region. Identity scoring is complete Only exact matches are counted, and the degree of similarity between amino acids is not considered. Only internal gaps are counted. The length is included, but gaps at the end of the array are not. Identity percentage = (number of matches × 100) ) / Length of the aligned region (with gaps)

[0037] The term "conservative amino acid substitution" refers to the following substitutions of amino acid residues within a group: 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 are substitutions of amino acids in proteins, such as side chains. Replace with an amino acid having a side chain with similar acidity, basicity, charge, polarity, or size. This allows for the preservation of protein activity.

[0038] The guide for substitution, insertion, or deletion is for the amino acid sequences of proteins from different species. Based on alignment, or based on multiple proteins having the same or similar function. It could be derived from a consensus sequence.

[0039] The term "vector" refers to a device used to transfer protein-coding information to host cells. Any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) It means...

[0040] The terms "expression vector" or "expression construct" are suitable for the transformation of host cells. and the expression of one or more heterogeneous coding regions manipulably linked to them (in conjunction with the host cell) This refers to a vector containing a nucleic acid sequence that directs and / or controls. Expression constructs are not limited to, but , affecting or controlling transcription and translation, and if introns are present, This may include sequences that affect RNA splicing of manipulably linked coding regions. .

[0041] The term "stimulus" refers to the stimulating molecule (e.g., the TCR / CD3 complex or CAR) and its This refers to the primary response induced by binding to a congener ligand (or tumor antigen in the case of CARs). And thereby, signaling via the TCR / CD3 complex, or Signaling via appropriate NK receptors or CAR signaling domains, etc. It mediates signaling events. A stimulus can mediate the altered expression of specific molecules. .

[0042] The term "stimulating molecule" is used in at least some aspects of immune cell signaling pathways. Therefore, immunotherapeutic cells provide cytoplasmic signaling sequences that regulate the activation of immune cells in a stimulating manner. This refers to molecules expressed by cells (e.g., T cells, NK cells, B cells). The signal is, for example, between the TCR / CD3 complex and the MHC molecule loaded with the peptide. It is a primary signal initiated by the binding of these, and includes proliferation, activation, differentiation, etc. This results in the mediation of T cell responses that are not limited to primary cytoplasmic signals acting by stimuli. The signaling sequence (also called the "primary signaling domain") is an immune receptor-activated tyrosine The present invention may contain a signaling motif known as a motif or ITAM. Examples of particularly useful ITAM-containing cytoplasmic signaling sequences include CD3 zeta and common Fc R-gamma (FCER1G), Fc-gamma RIIa, Fc-beta (Fc-epsilon R1b) ), CD3 Gamma, CD3 Delta, CD3 Epsilon, CD79a, CD79b, DAP This includes, but is not limited to, elements derived from 10 and DAP12.

[0043] The term "costimulatory molecule" refers to a molecule that specifically binds to a costimulatory ligand, thereby affecting T cells. Co-stimulatory responses by cells, for example, gene binding on T cells that mediate proliferation, etc. This refers to a co-stimulator. Co-stimulatory molecules are antigen receptors or their receptors that contribute to an efficient immune response. These are cell surface molecules other than Gand. Co-stimulatory molecules include 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 (C This includes, but is not limited to, D137.

[0044] The term "self" refers to any material originating from the same individual as the individual later reintroduced. .

[0045] When used herein, "intracellular signaling domain" refers to the intracellular portion of a molecule. It refers to the intracellular signaling domain, which is involved in the immune response of CAR-containing cells, such as CAR-T cells. It generates signals that promote effector function. For example, in CAR-T cells, immunoeffects Examples of receptor functions include helper activities such as cell lysis and cytokine secretion. It can be done.

[0046] As used herein, "immune effector cells" refers to immune response, for example, immune e This refers to cells involved in promoting the effector response. Examples of immune effector cells include T cells. For example, alpha / beta T cells and gamma / delta T cells, B cells, natural killer cells (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells It is included. [Modes for carrying out the invention]

[0047] An antibody that binds to the cell efflux pump ABCG2 is provided. Also, a product containing such an antibody or The code provides for pharmaceutical compositions, nucleic acids, recombinant expression vectors, cells, and kits. The presence or absence of ABCG2 expression and the level of ABCG2 expression in cells, such as tumor cells, can be detected. Methods for using antibodies to inhibit and / or ABCG2 function are also disclosed. In addition, cancer cells that express both ABCG2 and tumor-associated antigens (TAAs) on their cell surface. Multispecific antibodies, such as bispecific antibodies that bind to ABCG2, are provided. In addition, ABCG2 and A bispecific antibody is provided that binds to cancer cells expressing both efflux pumps ABCB1. In certain embodiments, the bispecific antibodies disclosed herein express both antigens. It binds to cells, but does not bind to cells that express only one of the antigens in a detectable manner. Furthermore, administering the anti-ABCG2 antibody or multispecific antibody disclosed herein to the target patient. Methods for treating cancer are provided, including the following.

[0048] Before describing the present invention in more detail, it should be noted that the present invention is not limited to the specific embodiments described. Therefore, please understand that this may change. The terms used in this specification are single. This is for the purpose of describing specific embodiments and is not intended to limit them. It should also be understood that the scope of the claims is limited only by the attached claims.

[0049] If a range of values ​​is provided, unless the context explicitly indicates otherwise, the upper and lower bounds of that range are... Each intervening value up to one-tenth of the lower limit unit between and and any other value within the stated range The values ​​or intervening values ​​are understood to be included within the present invention. These narrower ranges The upper and lower limits may be independently included in a narrower range, and any within the stated range Subject to certain excluded limitations, the scope described herein is included within this invention. If the scope includes one or both of the restrictions, the scope that excludes one or both of those restrictions is also This is included in the present invention.

[0050] Certain ranges are indicated herein by numerical values ​​preceded by the term “approximately”. The term "approximately" in this specification refers to the exact number that precedes it, as well as its usage. Provides literal support for numbers that are close to or approximately near the number preceding the word. Used to determine if a certain number is close to or approximately near a specific number listed. When determining this, a number that is close or roughly nearby but not listed is considered to be the one that is proposed. In the given context, it is a number that provides the substantial equivalent value of the numbers specifically listed. obtain.

[0051] Unless otherwise defined, all technical and scientific terms used herein are defined in accordance with the present invention. It has the same meaning as generally understood by those skilled in the art to which it belongs. Any method and materials similar or equivalent to those used in the implementation or testing of the present invention may also be used. While it is possible to do so, representative exemplary methods and materials are described here.

[0052] All publications and patents referenced herein are subject to the terms of each individual publication or patent. As is specifically and individually indicated, incorporated herein by reference To disclose and explain the methods and / or materials to which the publication is relatedly cited. Therefore, it is incorporated herein by reference. Citations of publications refer to their disclosures prior to the filing date. Therefore, it can be interpreted that the present invention acknowledges that there are no prior rights to such publications. It should not be interpreted. Furthermore, the date of the publication provided may differ from the actual publication date. It may need to be verified independently.

[0053] As used herein and in the appended claims, the singular forms "a", "an", and Note that "the" can refer to multiple things unless the context clearly indicates otherwise. Furthermore, it should be noted that the claims may be drafted to exclude optional elements. Therefore, this statement is "simply" related to the enumeration of elements of the claims. This can serve as a basis for the use of exclusive terms such as "only," or for the use of limitation by "excluding." It is intended to fulfill that role.

[0054] As will become apparent to those skilled in the art through reading this disclosure, the individual examples described and illustrated herein Each of these embodiments may be described in relation to several other embodiments without departing from the scope or spirit of the present invention. Individual components that can be easily separated from or combined with any of the features thereof It has elements and characteristics. Any enumerated method is possible in the order of the enumerated events, or logically. It can be executed in any other possible order.

[0055] Methods and compositions have been described with functional descriptions for grammatical fluidity or As will be explained below, the claims explicitly refer to 35 U.S.C. § 112(f) Unless otherwise formulated below, constructing a limitation of “means” or “steps” does not necessarily mean It should not be interpreted as being limited, but rather as being provided by the claims under the doctrine of equivalents. The definition and meaning and the full scope of the equivalents are given, and the claims are explicitly stated to be 35 US If it is formulated under §112(f) of the U.S.C., then 35 U.S.C. §112( It should be clearly understood that a perfect legal equivalent is given under f).

[0056] antibody As summarized above, this disclosure provides an antibody that binds to the cell efflux pump ABCG2. do.

[0057] ABCG2, also known as CD388 and BCRP, is an ATP-binding cassette subfacial. The Millie G member 2 (ABCG2) gene is expressed in a reduced amount in multidrug-resistant cells. It is an energy-dependent efflux pump responsible for drug accumulation.

[0058] In some embodiments, the antibodies disclosed herein are the extracellular domain of ABCG2. It binds to one or more sites. In a particular embodiment, the anti-ABCG2 antibody of this disclosure is human It binds to ABCG2. In certain embodiments, the anti-ABCG2 antibody of this disclosure binds to human cells. For example, it binds to human ABCG2 expressed on the cell surface of cancer cells. The antibodies disclosed herein have one or more extracellular domains (ECDs) of human ABCG2. Binding to the upper region, ECD may contain the sequence:KNDSTGIQNRAG(SEQ ID NO: 1). This sequence is the amino acid residue of the human ABCG2 sequence with registration number NP_004818. Corresponds to 417-428. In certain embodiments, the antibodies disclosed herein are human A The extracellular domain (ECD) of BCG2 binds to one or more sites, and the ECD has the sequence: LK This sequence may contain PKADAF (sequence number 2), and this sequence has registration number NP_004818. This corresponds to amino acid residues 499-506 of the human ABCG2 sequence. In a particular embodiment, The antibodies disclosed herein are those that have one or more extracellular domains (ECDs) of human ABCG2. The ECD binds to the site, and its sequence is: NLTTIASWLSWLQYFSIPRYGFTAL QHNEFLGQNFCPGLNATGNNPCNYATCTGEEYLVKQGIDL This sequence may contain SPWGLWKNH (sequence number 3), and this sequence has registration number NP_004818 This corresponds to amino acid residues 557-630 of the human ABCG2 sequence that possesses this characteristic.

[0059] The antibodies disclosed herein are i) Characteristics that inhibit emissions from ABCG2, ii) Increase the sensitivity of cancer cells to treatment with chemotherapeutic agents, thereby The property of reducing the IC50 of therapeutic agents by at least two times. iii) Properties that bind to human and cynomolgus monkey ABCG2, iv) Characteristics that are effective in in vivo cell killing assays, v) Properties that are effective in inhibiting tumor growth even in the absence of chemotherapy, vi) The property of preferentially binding to ABCG2 mutants constrained in an open three-dimensional structure. vii) Binds to cancer cells that express ABCG2 at higher levels compared to non-cancer cells. Furthermore, it binds significantly less to non-cancer cells, and has a lower binding range to ABCG2. Characteristics that have a harmonious relationship, and viii) The property of preferentially binding to ABCG2 mutants constrained by a closed three-dimensional structure. It may have one or more of the following.

[0060] In certain embodiments, the antibody of this disclosure has a lower EC50 than anti-ABCG2 antibody 5D3. It may have. When used herein, EC50 is half of the maximum response (e.g., maximum fluorescence). This refers to the concentration of the antibody that provides half of the strength. The antibodies in this disclosure are 100 nM or less, for example, 100nM~4nM, 80nM~4nM, 60nM~4nM, 40nM~4nM, 30n EC50 with M~4nM, 20nM~4nM, 15nM~4nM, or 10nM~4nM It may be possible. The EC50 of the test antibody is determined by flow cytometry or ELISA. It is possible. For example, flow cytometry can detect ABCG2 (e.g., human wild-type ABCG2). Cells expressing the mutant ABCG2) are subjected to an antibody in flow cytometry buffer (the antibody is The process involves contact with the continuously diluted solution and the antibody binding to the cell at room temperature or 4°C. This may involve incubation for a sufficient amount of time (e.g., 10 minutes to 1 hour). After incubation, the cells can optionally be washed to remove nonspecifically bound antibodies, and / Alternatively, cells may be brought into contact with a fluorescently labeled secondary antibody that specifically binds to the test antibody. After rinsing, the fluorescently labeled secondary antibody can be removed and the cells can be washed. The washed cells can then be used as a fluoroscopy solution. Cells can be sorted by tubometry and the number of cells bound to a fluorescently labeled secondary antibody can be counted. The concentration that provides half of the maximum response (e.g., half of the maximum fluorescence intensity) is measured as EC50. It is determined. In variations of the flow cytometry assay, cells are determined to be ABCG2 This may be an overexpressing 293T cell. In certain embodiments, the antibody of this disclosure is anti-ABC. It may have a higher EC50 than the G2 antibody 5D3.

[0061] In a particular embodiment, having one or more of the characteristics i) to viii) listed above In addition, one or more of the anti-ABCG2 antibodies of this disclosure are EC of anti-ABCG2 antibody 5D3. It may have an EC50 which is at least half of 50. In certain embodiments, the following are listed above. In addition to having one or more of the characteristics i) to viiii), the anti-ABCG2 antibody of this disclosure One or more of these have an EC50 that is at least twice that of anti-ABCG2 antibody 5D3. It is possible to have.

[0062] The IC50 of a test antibody can be determined by measuring its inhibition of cell proliferation. 0 is the value of the antibody that produced half of the maximum response, measured by using the test antibody alone. The concentration can be determined. The IC50 of the chemotherapeutic agent is measured in the absence and presence of the test antibody. The effect of antibodies against IC50 chemotherapeutic agents can be determined. The chemotherapeutic agent is topotecan. To obtain. The cells may be cancer cell lines. Cancer cell lines include human acute lymphoblastic leukemia (A). N6 / is a doxorubicin-selective B1-positive variant of the LL cell line NALM6. ADR is possible. N6 / ADR cells are also called NALM6 / ADR cells. Antibodies When determining IC50, cells may be contacted with the antibody alone, and the antibody is tested in serial dilutions. The effect of the antibody on the IC50 of the drug was determined by contacting cells with the antibody and the chemotherapeutic agent. The drug is then tested in serial dilutions. Cells are kept at 37°C for a set period of time (e.g., 24 hours to 84 hours). The cells can be incubated and their viability evaluated using standard reagents and methods. The antibodies disclosed in this specification increase the sensitivity of cancer cells to treatment with chemotherapeutic agents. This reduces the IC50 of chemotherapy agents by at least five times. Cancer cells are N6 / ADR may be. The chemotherapeutic agent may be topotecan. In a particular aspect, this The antibodies shown increase the IC50 of the chemotherapy agent by 5 times or more, for example, 6 times or more, 7 times or more, 8 times or more. It can reduce the value by 9 times or more, or even 10 times or more, for example, by 5 to 10 times.

[0063] In certain embodiments, one or more of the anti-ABCG2 antibodies disclosed herein are used in humans and It binds to both ABCG2 in cynomolgus monkeys. This characteristic is beneficial for antibody stability in animal models. It can be used to determine totality.

[0064] In certain embodiments, the anti-ABCG2 antibody disclosed herein is specific to ABCG2 It is not a target antigen and does not show significant binding to other antigens.

[0065] In a particular embodiment, the in vitro cell-killing activity of the antibodies of this disclosure is observed with the 5D3 antibody. It may be superior to [the other]. For example, the antibody of this disclosure has more than twice the in vitro [result] of the 5D3 antibody. It may possess cell-killing activity.

[0066] In certain embodiments, one or more of the antibodies provided herein have an open three-dimensional structure. It preferentially binds to the ABCG2 mutant that is constrained by E211. This could be human or cynomolgus monkey ABCG2 containing Q, where amino acid position numbering is H This refers to ABCG2. Such antibodies are constrained by a closed three-dimensional structure and are wild-type ABC Compared to the G2 or ABCG2 mutant, the ABCG2 mutant, which is constrained in an open three-dimensional structure, At least twice (for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more) It can bind with the affinity shown above.

[0067] In certain embodiments, one or more of the antibodies provided herein have a closed three-dimensional structure. It preferentially binds to ABCG2 mutants that are constrained by: (i) (ii) K86M and S87A, (ii) K86M, S87A, and Q126A, or (iii) K86M, S87A, Q126A, R246E (The amino acid position numbering is based on human ABC) This may include human or cynomolgus monkey ABCG2 (referring to G2). Such antibodies Compared to wild-type ABCG2 or ABCG2 mutants constrained in an open three-dimensional structure, closed The ABCG2 mutant constrained by the resulting three-dimensional structure exhibits at least twofold (e.g., twofold, threefold, fourfold) It can bind with affinity (times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more).

[0068] In certain embodiments, the antibodies provided herein are monospecific bivalent anti-ABCG2 antibodies. In a particular embodiment, the monospecific bivalent anti-ABCG2 antibody of this disclosure is anti-ABCG At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 are present in antibody 5D3, It does not contain all 11 or 12 of the HCDR1-3 and LCDR1-3.

[0069] The 5D3 antibody has the sequence: QVQLQESGPGLVKPSQSLSLTCTVTGFSI TSDYAWNWIRQFPGKKLEWMGYINFDGGTTYNPSLRGRIS ITRDTSKNQFFLQLRSVTPEDTATYYCATFYGAKGTLDYW Variable heavy chain having GQGTSVTVSS (Sequence ID 4), and array: DIVLTQSPSSFSVSLGDRVTISCKASGYILNRLA WYQQKKPGNAPRLLISGATSLETGFPSRFSGTGSGKDYTLS ISSLQTEDVGTYYCQQYWSTPWTFGGGTKLEIK(Sequence ID 5) Includes a variable light chain having

[0070] HCDR1-3 and LCDR1-3 of 5D3 antibodies as defined according to Kabat nomenclature. The following applies: HCDR 1: GFSITSDYAW (Sequence ID 6); HCDR 2: GYINF DGGTTYNPSLRG(Sequence ID 7);HCDR 3: ATFYGAKGTLDY (Sequence ID 8); LCDR 1: KASGYILNRLA (Sequence ID 9); LCDR 2: GATS LET(sequence number 10);LCDR 3: QQYWSTPWT(sequence number 11).

[0071] In some embodiments, one or more of the target antibodies express ABCG2. When bound to cells, it may interfere with the function of the cellular ABCG2 protein. Therefore, the present disclosure One or more antibodies may inhibit efflux by the ABCG2 protein, for example, the non- Compared to emissions from ABCG2 in the presence of ABCG2, emissions are 5% or more, for example, 10% or less. Top, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60 It is reduced by more than %, more than 70%, more than 80%, or more than 90%. In some embodiments Furthermore, when the target antibody binds to cells expressing ABCG2, it triggers other mechanisms, for example... This makes ABCG2 leaky, which in turn hinders the uptake of chemotherapy agents. By enhancing and / or reducing cell viability, the action of ABCG2 is inhibited. obtain.

[0072] In a particular embodiment, an antibody that binds to ABCG2 is provided, and the antibody is compatible with ABCG2. Regarding binding, the variable heavy chain (VH) region and variable light chain (VL) region of the antibodies listed in Table 2 and An antibody containing a heavy chain complementarity determining region (HCDR) and a light chain CDR (LCDR) in each pair. It competes with ABCG2. For example, in one embodiment, the anti-ABCG2 antibody of this disclosure has a binding relationship with ABCG2. In terms of compatibility, it competes with the G2.302 antibody listed in Table 2. In certain embodiments, HC DR1-3 and LCDR1-3 are defined according to Kabat nomenclature.

[0073] In certain embodiments, the antibody is the HCDR1, HCD of the VH region of the antibodies listed in Table 2. Includes R2 and HCDR3. In a particular aspect, HCDR1, HCDR2, and HC DR3 is defined according to Kabat nomenclature. For example, in one embodiment, ABCG2 Regarding binding, the anti-ABCG2 antibody of this disclosure competes with the G2.302 antibody listed in Table 2. The body contains HCDR1, HCDR2, and HCDR3 of the VH region of the G2.302 antibody.

[0074] The task of determining whether the first antibody competes with the second antibody for binding to ABCG2. A preferred approach may be used. The first antibody binds to the compound, while the second antibody Whether it "competes" with the body can be easily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, through antibody competition assays. The antibody sample can be bound to a solid support. Then, it competes with the first antibody. A sample of a second antibody, which is thought to be capable of doing so, is added. One of the two antibodies is Labeled. Labeled and unlabeled antibodies bind to separate sites on the compound. Combined and labeled antibodies are at the same level, regardless of whether or not there are competing antibodies present. They bind. However, if the interaction sites are the same or overlap, the unlabeled antibody competes. The amount of labeled antibody that binds to the antigen decreases. If there is an excess of unlabeled antibody, the bound antibody Even then, only a very small amount of labeled antibody binds.

[0075] For the purposes of this disclosure, the competing antibody binds to the compound by approximately 30% or more, and approximately 40% or more. % or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 85% or more, approximately This reduces by 90% or more, approximately 95% or more, or approximately 99% or more. Details of the procedure for carrying out (i) are well known in the art, for example, Harl ow and Lane,Antibodies,A Laboratory Manu al,Cold Spring Harbor Laboratory Press,C old Spring Harbor,New York,1988,567-569, This can be found in 1988, ISBN 0-87969-314-2. (i) can be quantified by using purified antibodies. The standard curve is 1 This can be established by measuring the titer of one antibody against itself, that is, the same antibody However, it is used for both labeling and as a competitor. It inhibits the binding of the labeled antibody to the target epitope. The titer of the unlabeled competitive antibody is measured. The results are plotted to determine the desired degree of binding inhibition. The concentrations required to achieve this can be compared.

[0076] In a particular embodiment, antibodies that specifically bind to ABCG2 are (i) listed in Table 2. The HCDR1-3 and light chain C are pairs of variable heavy chain (VH) and variable light chain (VL) regions of antibodies. DR (LCDR1-3), (ii) HCDR1-3 of the VH region of antibodies listed in Table 2, Or (iii) containing LCDR1-3 of the VH region of the antibodies listed in Table 2. HCDR and LCDR can be defined based on Kabat nomenclature.

[0077] In certain embodiments, the antibodies of this disclosure that specifically bind to human ABCG2 are listed in Table 2. The HCDR1, HCDR2, and HCDR3 sequences of the antibodies, as well as LCDR1 and LCD It contains R2 and LCDR3 sequences. In addition to binding to human ABCG2, it is specified herein One or more of the antibodies provided are from other mammalian species, such as mice, monkeys, and chimpanzees. —Can bind to ABCG2 from sources such as these. Antibodies can be produced in mice or rats. Table 2 The animals that produced the antibodies are shown. Some of the antibodies were humanized. Table 2: From left to right, Column 1: Anti-ABCG2 antibody name, Column 2: VH region, Column 3: HCD R1, 4th row: HCDR2, 5th row: HCDR3, 6th row: VL area, 7th row: LCDR Rows 1 and 8: LCDR2, Row 9: LCDR3. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

[0078] The anti-ABCG2 antibodies listed in Table 2, also called anti-KPG2 antibodies, can be referred to by the antibody numbers listed in Table 2. The antibodies can be referred to by the antibody numbers. [[ID=7​The term "antibody molecule" encompasses antibodies as defined herein, and their antigen-binding fragments. Includes. In certain embodiments, the antibody molecule has two variable light chains (VLs) and two variable heavy chains ( It includes (VH). In certain embodiments, the antibody molecule further includes heavy chain and light chain constant regions. The heavy and light chain constant regions may be derived from human antibodies, such as human IgG1 antibodies. The constant region of the gG1 heavy chain (HC) is a mutation that reduces antibody-dependent cell-mediated cytotoxicity (ADCC). It may be modified to include, or alternatively, two VH chains each contain different human Ig The G1 HC constant region can be conjugated, and each human IgG1 HC constant region is different. This human IgG1 has substitutions that are convenient for dimer formation between the constant regions of HC. The C region is described in further detail herein. In certain embodiments, the antibody molecule is bispecific In the case of a sex antibody molecule, one of the constant regions of human IgG1 HC has a positively charged side chain. The substitution may include introducing one or more amino acids, and the other human IgG1 HC constant region may include 2 One or more amino acids having load side chains that are favorable for dimer formation between different HCs This may include the substitutions to be introduced.

[0080] In certain embodiments, antibody molecules have pairs of the VH and VL regions of the antibody listed in Table 2. It contains HCDR1-3 and / or LCDR1-3. In one embodiment, the antibody molecule is shown in Table 2 Includes HCDR1-3 and / or LCDR1-3 of the G2.248 antibodies listed below. In this case, the antibody molecules are the G2.255 antibodies HCDR1-3 and / or listed in Table 2. This includes LCDR1-3. In one embodiment, the antibody molecule is G2.256 listed in Table 2. The antibody comprises HCDR1-3 and / or LCDR1-3. In one embodiment, the antibody molecule is This includes the G2.65 antibodies HCDR1-3 and / or LCDR1-3 listed in Table 2. In this embodiment, the antibody molecule is HCDR1-3 and / or G2.30 antibodies listed in Table 2. This includes LCDR1-3. In one embodiment, the antibody molecule is G2.173 listed in Table 2. The antibody comprises HCDR1-3 and / or LCDR1-3. In one embodiment, the antibody molecule is Table 2 lists the G2.333 antibody, G2.343 antibody, G2.636 antibody, and G2.62 HCDR1-3 antibodies of 9, G2.643, G2.420, or G2.631 and / or LCDR1-3. In one embodiment, the antibody molecule is G2 listed in Table 2. The .636 antibody comprises HCDR1-3 and / or LCDR1-3. In one embodiment, the antibody The molecules are HCDR1-3 and / or LCDR1-3 of the G2.318 antibody listed in Table 2. Includes.

[0081] In certain embodiments, the antibody has a pair of H regions in the VH and VL regions of the antibody listed in Table 2. Includes CDR1-3 and / or LCDR1-3, and increases the IC50 of the chemotherapeutic agent by 5 times or more, or by 6 times. More than 7 times, more than 8 times, more than 9 times, or more than 10 times, for example, reducing by 5 to 10 times ru.

[0082] EC50 may be the antibody concentration that produces the 50% maximum response (for example, the response is due to the antibody and (This is binding to the antigen). In one embodiment, the antibody has an EC50 of 100 nM or less and / or It has an EC50 that is at least half the EC50 of anti-ABCG2 antibodies such as 5D3, and table Includes HCDR1-3 and / or LCDR1-3 of the G2.248 antibodies listed in 2. In this embodiment, the antibody is an anti-ABCG2 such as EC50 and / or 5D3 with an EC of 100 nM or less. The antibody has an EC50 that is at least half of the antibody's EC50, and is listed in Table 2 as G2.255. The antibody comprises HCDR1-3 and / or LCDR1-3. In one embodiment, the antibody is 10 EC50 of 0 nM or less and / or EC50 of anti-ABCG2 antibodies such as 5D3 Having half the EC50, the G2.256 antibodies HCDR1-3 and / Or it contains LCDR1-3. In one embodiment, the antibody has an EC50 of 100 nM or less and / Alternatively, it has an EC50 that is at least half the EC50 of an anti-ABCG2 antibody such as 5D3, This includes the G2.65 antibodies HCDR1-3 and / or LCDR1-3 listed in Table 2. In this embodiment, the antibody is an anti-ABCG2 such as EC50 and / or 5D3 with an EC of 100 nM or less. The G2.30 antibody has an EC50 that is at least half of the antibody's EC50 and is listed in Table 2. The body contains HCDR1-3 and / or LCDR1-3. In one aspect, the antibody is 100 EC50 of nM or less and / or at least half the EC50 of anti-ABCG2 antibodies such as 5D3 It has an EC50 of the G2.173 antibodies listed in Table 2, HCDR1-3 and / or This includes LCDR1-3.

[0083] In some embodiments, the antibody has a VL region and V region present in a separate polypeptide. In other embodiments, the H region is included, and the VL and VH regions are within a single polypeptide. It is included in.

[0084] The antibodies of this disclosure may include humanized light chains, humanized heavy chains, or both, in certain embodiments. The antibodies are listed in Table 2 as G2.173 Humanized 1, G2.173 Humanized 1, or G2.173 Human It may be a humanized antibody containing a VH region as shown for antibody 1. The antibodies are listed in Table 2 as G2.173 Humanized 1, G2.173 Humanized 1, or G2.173 It may be a humanized antibody containing a VL region, as shown for humanized antibody 1. Specific embodiment In this case, the antibodies are listed in Table 2 as G2.173 Humanized 1, G2.173 Humanized 1, or G2.1 It can be a humanized antibody containing the VH and VL regions as shown for 73 Humanized 1 antibody. .

[0085] The antibody disclosed herein consists of an Ig monomer, a Fab fragment, an F(ab')2 fragment, an Fd fragment, and scF The group can be selected from v, scAb, dAb, and Fv.

[0086] multispecific antibodies In certain embodiments, the antibodies of this disclosure are present in epithelial cells of two different target proteins. It is multispecific and can bind to different antibodies. The number of target proteins, and therefore the number of different epitopes, can vary, with two (i.e.) It can be bispecific, three (triple specific), four, or more.

[0087] In certain embodiments, the antibodies of this disclosure bind to at least two different epitopes. It is a multispecific (e.g., bispecific) antibody that can perform such actions, and one of the epitopes is It is located in ABCG2 (for example, human ABCG2), while other epitopes are found on the cell surface of cancer cells. It is found in tumor-associated antigens (TAAs) expressed on the surface. In another embodiment, the multispecificity of the present disclosure The antibody binds to human ABCG2 and the efflux pump MDR-1. In a particular embodiment, The VH and VL chains of the disclosed bispecific antibody are such that the antibody expresses both antigens in cells, for example. However, while it binds to cancer cells, its effectiveness against cells expressing only one of the antigens is substantially reduced. Selected to indicate a join.

[0088] In certain embodiments, the VH and VL chains of the bispecific antibody of this disclosure are such that the antibody is both It binds to cells that overexpress the antigen, and either expresses both antigens at normal levels or one of the antigens Cells that express or overexpress only one of these genes are selected to show substantially lower binding. These antibodies specifically bind to cancer cells that overexpress both antigens, and therefore... Therefore, it has minimal off-target effects due to reduced binding to normal cells. When used, the term "overexpressed" refers to an expression level higher than what is detected in normal cells. It means encompassing levels. For example, cancer cells that overexpress TAA are considered to be cancer Higher levels than in normal cells of the same type, such as epithelial cells. It expresses TAA. Normal cells either do not express TAA or express a certain level of TAA. It can be expressed. Cancer cells express TAA at levels compared to normal cells of the same type. It can be overexpressed.

[0089] Tumor-associated antigens (also referred to herein as cancer-associated antigens) are found in cancer cells, This refers to antigens that are overexpressed compared to their expression levels in non-cancer cells of the same type. For example, TAA is not expressed at detectable levels in normal cells, but is expressed in cancer cells. Antigens are the same cell type as normal cells and cancer cells, for example, epithelial cells. In another embodiment, TAA is expressed in normal cells but at higher levels in cancer cells. It is an expressed antigen. TAA can be expressed on the cell surface of mammalian cancer cells. In one embodiment, TAA is CD47, PDL1, erbB-1, erbB-2, or EG It may be FR. Tumor-associated antigens may be neoantigens. Neoantigens are encoded by tan. The amino acid sequence of the protein is altered by comparing it with the amino acid sequence of the unmutated protein, which is a tumor-specific method. This is a class of tumor antigens that arise from heterologous mutations.

[0090] In a particular embodiment, the bispecific antibody is used for ABCG2 and the efflux pump MDR-1 or cancer It specifically binds to cancer cells that overexpress both the related antigen and the associated antigen. In a particular embodiment, two The heavily specific antibody binds to cancer cells that overexpress both ABCG2 and MDR-1, and A For cells that do not overexpress both BCG2 and MDR-1, substantially lower binding is observed. show.

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

[0092] In certain embodiments, the bispecific antibodies of this disclosure are ABCG2 and TAA or MDR-1 Both are expressed at more than twice the level of normal cells (for example, at least three times, four times). It can be selected based on its binding to cells expressing at levels of 2x, 5x, 10x or more. , normal cells, or overexpression of only one of ABCG2 and TAA or MDR-1 For cells that do not bind to this cell type, it exhibits substantially lower binding.

[0093] In a particular embodiment, bispecific antibodies are sensitive to cancer cells in response to treatment with chemotherapeutic agents. It increases receptivity, thereby allowing the chemotherapy agent to be administered together with multispecific antibodies. IC50 is the I of the chemotherapeutic agent when administered together with an anti-ABCG2 monospecific bivalent antibody. It reduces IC50 by at least twice compared to C50. IC50 is as provided herein. It can be measured by method. The chemotherapy agent may be topotecan. Cancer cells are drug resistant. These may be cancer cells. In certain embodiments, the multispecific antibodies of this disclosure are chemotherapeutic agents. The IC50 is multiplied by 5 or more, for example, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. For example, it can be reduced by 5 to 10 times.

[0094] In certain embodiments, bispecific antibodies are present in the absence of administration of chemotecan or other chemotherapy agents. Even under these conditions, it may exhibit in vivo cell-killing activity, such as a reduction in tumor volume.

[0095] In some embodiments, the multispecific antibodies of this disclosure, for example, bispecific antibodies, It may include a common light chain. As used herein, the term "common light chain" means a general light chain. This refers to the use and incorporation of two copies of the same light chain into multispecific antibodies. If so, the light chain in the assembled multispecific antibody associates with the ABCG2-specific heavy chain, A second copy of the same light chain associates with either a TAA-specific heavy chain or an MDR-1 antigen-specific heavy chain. The common light chain has a VL chain containing, for example, LCDR1-3 of the antibodies listed in Table 2. Antibodies, e.g., G2.248, G2.255, G2.256, G2.65, G2.302 , G2.173, G2.173.Humanized 1, G2.173.Humanized 2, or G2.173 This may be derived from anti-ABCG2 antibodies such as humanized 3 antibodies. In other embodiments, common The light chain may be derived from an anti-MDR-1 antibody such as MRK16 or 15D3. In one embodiment, the common light chain is an unrelated antibody, antibody library, or synthetically designed It could be from a source of antibodies produced in vitro. Therefore, bispecific antibodies do not contain a common light chain, but instead have a first heavy chain that binds to G2 and It may include a first light chain, as well as a second heavy chain and a second light chain that bind to another antigen.

[0096] Bispecific antibodies against ABCG2 and MDR1 In a particular embodiment, the bispecific antibody molecules that bind to ABCG2 and MDR1 are two It may include the same variable light (VL) chain, a first variable heavy (VH) chain, and a second VH chain, VL Each chain contains an antigen-binding site for MDR1, and the first VH chain is an antigen-binding site for MDR1. The second VH chain contains the protobinding site, and the second VH chain contains the antigen-binding site for ABCG2. The H chain binds to ABCG2 when it pairs with one of the light chains. The second VH chain contains HCDR1-3 of the VH chains of the anti-G2 antibodies listed in Table 2. The second VH chain contains HCDR1-3 of the VH chain of anti-MDR1 antibodies such as 15D3, and is common. The light chain is the VL region of another anti-MDR1 antibody, such as anti-MDR1 antibody 15D3 or MRK16. Includes LCDR1-3.

[0097] In a particular embodiment, the first VH chain of a bispecific antibody is an anti-MDR1 antibody such as 15D3. It contains HCDR1-3 of the VH chain from the body, and HCDR1 has the sequence: GFTFSRYTMS( HCDR2, which includes sequence number 419, has the sequence: VATISSGGGNTYYPDSVK G (Sequence ID 362), VATISSGGGQTYYPDSVKG (Sequence ID 363), Or containing VATISSGGGSTYYPDSVKG (SEQ ID NO: 364), HCDR3 is , including sequence:ARYGAGDAWFAY (sequence number 365). In a particular embodiment, two The second VH chain of the hemispecific antibody is the VH chain of the anti-ABCG2 antibody having the sequence shown in Table 2. It includes HCDR1-3. In a particular embodiment, the common VL chain of the bispecific antibody is anti-M This contains LCDR1-3 of the VL chain of DR1 antibody 15D3.

[0098] In certain embodiments, the second VH chain of the bispecific antibody is anti-ABCG as listed in Table 2. It contains HCDR1-3 of the VH chain of two antibodies, G2.255. In a particular embodiment, the second The VH chain has the following amino acid sequence: EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQ TPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTL YLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVS (Sequence ID 366) at least 90%, at least 95%, at least 99%, Or it contains an amino acid sequence that is 100% identical.

[0099] In a particular embodiment, two identical VL chains have the amino acid sequence: DVLMTQTPLSL PVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKL LIYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVY Anti-MDR1 inhibitor containing YCFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367) Contains LCDR1-3 of the VL chain of body 15D3.

[0100] In a particular embodiment, two identical VL chains contain LCDR1-3 of the anti-MDR1 antibody. , (i) LCDR1 contains the sequence: RSSQSIVHSTGNTYLE (SEQ ID NO: 368) and (ii) LCDR2 contains the sequence: KVSNRFS (SEQ ID NO: 305), (iii) LCDR3 contains the sequence: QGSHFPRT (SEQ ID NO: 369).

[0101] In certain embodiments, two identical VL chains have the amino acid sequence: DVLMTQTPVSL SVSLGDQASISCRSSQSIVHSTGX 2 TYLEWYLQKPGQSPK LLIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YYCFQASHFPRTFGGGTKLEIK (SEQ ID NO: 370) (where X 2 is N , Q, or S) and contain the LCDR1-3 of the VL chain of an anti-MDR1 antibody.

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

[0103] In certain embodiments, the subject antibody contains a VH chain containing the HCDR of the VH chain listed in Table 2 ( e.g., the G2.255 antibody), and the LCDR of the MRK16 antibody or 15D3 antibody, or its humanized form, and may contain a VL chain. The HCDR and LCDR are according to the Kabat nomenclature Therefore, it can be defined as follows:

[0104] The amino acid sequence of the VL chain of the MRK16 antibody is as follows: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGNTYLE WYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTLK ISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK(Sequence No. 37) 4)

[0105] The amino acid sequence of the humanized VL chain of the MRK16 antibody is as follows: DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSTGX 2 TYL EWYQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTL KISRVEAEDVGVYYCFQASHFPRTFGGGTKLEIKR(Array code) 375)(X here) 2 (This is N, Q, or S)

[0106] The VL strands LCDR1-3 have the following sequence: CDR1(RSSQSIVHSTGX 2 TYL EW, Sequence ID 376) (where X 2 (is N, Q, or S), CDR2 (KISN RFSG (SEQ ID NO: 377), and CDR3 (FQASHFPRTF, SEQ ID NO: 378) It may have.

[0107] In a particular embodiment, the VL chain is MRK16 VL chain sequence: DVLMTQTPVSL SVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKL LIYKISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQASHFPRTFGGGTKLEIK (sequence number 374), or array:DIVM TQTPLSSPVTLGQPASISCRSSQSIVHSTGX 2 TYLEWYQQ RPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRV EAEDVGVYYCFQASHFPRTFGGGTKLEIKR(Sequence ID 375)( Here X 2 This sequence has at least N, Q, or S, and is a humanized form of this sequence. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or higher (including 100%) It may have a unified sequence.

[0108] In a particular embodiment, LCDR is a 15D3 VL chain: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLE WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRLEAEDLGVYYCFQGSHFPRTFGGGTRLEIK(Sequence No. 36) It is from 7).

[0109] The 15D3 LCDR1~3 array, defined according to Kabat nomenclature, is as follows: be. 15D3 LCDR1: RSSQSIVHSTGNTYLE (Sequence ID 368) 15D3 LCDR2: KVSNRFS (Sequence ID 305) 15D3 LCDR3: QGSHFPRT (Sequence ID 369)

[0110] In a particular embodiment, the VL chain is the 15D3 VL chain sequence: DVLMTQTPLSLP VSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYY At least 85 for CFQGSHFPRTFGGGTRLEIK (SEQ ID NO: 367) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 Identity of %, 96%, 97%, 98%, 99%, or higher (including 100%). It may have an array.

[0111] In a specific embodiment, the target antibody is a VL chain containing the LCDR of the VL chain listed in Table 2. and VH chains containing HCDRs of MRK16 antibody or 15D3 antibody or their humanized forms. It is possible. HCDR and LCDR can be defined according to Kabat nomenclature.

[0112] The amino acid sequence of the VH chain of the MRK16 antibody is as follows: EVILVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQ TPEKRLEWVATISSGGGNTYYPDSVKGRFTISRDNAKNNL YLQMSSLRSEDTALYYCARYYRYEAWFASWGQGTLVTVSA (Sequence ID 379)

[0113] The amino acid sequence of the VH chain of the 15D3 antibody is as follows: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQ TPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence ID 380)

[0114] The amino acid sequence of the humanized VH chain of the 15D3 antibody is as follows: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQ APGKGLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNSKNS LYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVS SA (Sequence No. 381) (where X 2 (This is N, Q, or S)

[0115] The 15D3 HCDR1~3 sequences, defined according to Kabat nomenclature, are as follows: be. 15D3 HCDR1: RYTMS (Sequence ID 382), 15D3 HCDR2: TISSGGGX 2 TYYPDSVKG (Sequence ID 383) (X here) 2 (is N, Q, or S), 15D3 HCDR3: YGAGDAWFAY (Sequence ID 384)

[0116] In a particular embodiment, the VH chain has the 15D3 VH chain sequence: EVKVVESGGVLVRPGGSLKLSCAASGFTFSRYTMSWVRQ TPEKRLEWVATISSGGGNTYYPDSVKGRFTVSRDNAMSSL YLQMSSLRSEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence ID 380), or array: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQ APGKGLEWVATISSGGGX 2 TYYPDSVKGRFTVSRDNSKNS LYLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVS SA (Sequence No. 381) (where X2 This sequence has N, Q, or S Humanized form and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more ( It may have sequences that are identical (including 100%).

[0117] In certain embodiments, the second VH chain includes a humanized form of the VH chain of the antibody listed in Table 2. Furthermore, the second VH chain contains the humanized form of the VH chain of the anti-MDR1 antibody 15D3, and humanized 15D 3. The VH chain has the following sequence: EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQ APGKGLEWVATISSGGGNTYYPDSVKGRFTVSRDNSKNSL YLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence ID 385) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQ APGKGLEWVATISSGGGQTYYPDSVKGRFTVSRDNSKNSL YLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (Sequence ID 386), or EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYTMSWVRQ APGKGLEWVATISSGGGSTYYPDSVKGRFTVSRDNSKNSL YLQMNSLRTEDTALYYCARYGAGDAWFAYWGQGTLVTVSS (SEQ ID NO: 387) has a common light chain, and the LCD of the VL region of anti-MDR1 antibody 15D3 Includes R1-R3.

[0118] In a specific embodiment, the bispecific antibody binds to MDR1 and ABCG2, and in chemotherapy The IC50 of the agent should be more than twice as high (for example, 3, 4, 5, 6, 7, 8, 9, 10 times, or more). ) reduces the common light chain, the VH chain containing HCDR from the VH chain listed in Table 2, and 1 It contains VH chains containing HCDR from anti-MDR1 antibodies such as 5D3. The common light chain is shown in Table 2. From the enumerated VL chains (for example, the VL of the antibody in Table 2 from which VH HCDR originates), Alternatively, it may contain LCDR from anti-MDR1 antibodies such as MRK16 or 15D3. In this embodiment, the chemotherapeutic agent is topotecan.

[0119] Bispecific antibodies against ABCG2 and TAA Furthermore, bispecific antibodies having at least one of the above characteristics are described herein. Provided. In certain embodiments, the bispecific antibody molecule of this disclosure is provided as an ATP-bound cassette. It binds to family G member 2 (ABCG2) and tumor-associated antigen (TAA), and antibody The child includes two identical variable light (VL) chains, a first variable heavy (VH) chain, and a second VH chain. Each VL chain contains an antigen-binding site for ABCG2, and the first VH chain contains ABCG The second VH chain contains an antigen-binding site for 2, and the second VH chain contains an antigen-binding site for TAA, The second VH chain either binds to the TAA when paired with one of the light chains, or to the VL chain. Each contains an antigen-binding site for TAA, and the first VH chain contains an antigen for ABCG2. The second VH chain contains a binding site, and the first VH chain contains an antigen-binding site for TAA, and the second VH chain contains an antigen-binding site for TAA, and the first VH chain is It binds to ABCG2 when paired with one of the light chains. TAA is CD47, P It could be DL1, EGFR, erbB-1, or erbB-2.

[0120] In a particular embodiment, each VL chain contains an antigen-binding site for ABCG2, and the first The VH chain contains an antigen-binding site for ABCG2, and the second VH chain is an anti-TAA Including the primordial binding site, the second VH chain binds to the TAA when it pairs with one of the light chains. do.

[0121] In certain embodiments, the first VH chain is the heavy chain complement of the anti-ABCG2 antibody listed in Table 2. Includes sex determination regions 1-3 (HCDR1-3).

[0122] In a particular embodiment, the first VH chain has heavy chain complementarity determining regions 1-3 (HCDR1-3) HCDR1 contains sequence:DDYVH (sequence number 85), and HCDR2 contains sequence :Includes RIDPANGNTRYAPKFRG (Sequence ID 115), and HCDR3 is sequence :Contains PLWVGGFAY (SEQ ID NO: 157), or the first VH chain contains amino acids array: QVQLQQSGADLVRPGASVKLSCTASGFNIKDDYVHWVKQ RPEQGLEWIGRIDPANGNTRYAPKFRGKATMTADTSSNTA YLQLSSLTSADTAVYYCSPPLWVGGFAYWGQGTLVTVSS( Sequence ID 16), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQ APGQGLEWIGRIDPANGNTRYAPKFRGRATMTADTSISTA YMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS( Sequence ID 17), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQ APGQGLEWIGRIDPAQGNTRYAPKFRGRATMTADTSISTA YMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS( Sequence ID 18), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQ APGQGLEWIGRIDPASGNTRYAPKFRGRATMTADTSISTA YMELSRLRSDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS( For sequence number 19), at least 90%, at least 95%, or 100% identical It contains an amino acid sequence that is of sex.

[0123] In some embodiments, the antigen-binding sites of the two VL chains are the lighter antibodies listed in Table 2. Includes chains CDR1-3 (LCDR1-3).

[0124] In some embodiments, the antigen-binding sites of the two VL chains are in the sequence: RSSQSLVHS LCDR1 containing DVNTYLH (sequence number 270), sequence: KVSNRFS (sequence number 270) LCDR2 containing 305), and L containing sequence:SQTTHVPYT(sequence number 334) The CDR3 or VL chain has the following amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDVNTYLH WYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRVESEDLGIYFCSQTTHVPYTFGGGTKLEIK(Sequence ID 19) 9), or DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDVNTYLH WYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRVEAEDVGVYFCSQTTHVPYTFGGGTKLEIK(Sequence ID 20 A that has at least 90%, at least 95%, or 100% identity with respect to 0). Contains a mino acid sequence.

[0125] In a particular embodiment, the first VH chain has heavy chain complementarity determining regions 1-3 (HCDR1-3) HCDR1 contains sequence:SGYIS, and HCDR2 contains sequence:WIYAGTG ISNFNQKFTG is included, and HCDR3 contains the sequence:GARKTLDF, or the The VH chain of 1 has the following amino acid sequence: QGQMHQSGAELVKPGASVKLSCKTSGFTFNSGYISWLKQ KPRQSLEWIAWIYAGTGISNFNQKFTGKAQLTVDTSSSTA YMQLSSLTSADSAIYFCASGARKTLDFWGQGTSVTVSS(distribution For column number 15), at least 90%, at least 95%, or 100% identity. It contains the following amino acid sequence.

[0126] In certain embodiments, the antigen-binding sites of the two VL chains are the light chain C of the antibodies listed in Table 2. Includes DR1-3 (LCDR1-3).

[0127] In a particular embodiment, the antigen-binding sites of the two VL chains are sequence: G2.65 KASDQ LCDR1 containing INYWLA (sequence number 269), array: GATSLET (sequence number 1 LCDR2 containing 0), and LCD containing array: QQYWTTPYT (sequence number 333) The R3 group or VL chain has the following amino acid sequence: DIQMTQSSSYLSVSVGGRVTITCKASDQINYWLAWYQQK PGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSFQ For TEDVATYYCQQYWTTPYTFGGGTKVEIK (Sequence ID 198) an amino acid sequence that is at least 90%, at least 95%, or 100% identical Includes.

[0128] In some embodiments, TAA is EGFR, and the second VH chain is an amino acid sequence :QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWI RQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQ FSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTV The heavy chain complementarity determination region 1-3(H) of the VH chain of the 6B3S antibody, including SS (SEQ ID NO: 388). Includes CDR1~3)

[0129] In certain embodiments, bispecific antibodies are HCDR1-3, as shown in the table below. A first VH chain containing HCDR1-3, a second VH chain containing HCDR1-3, and a co- Includes common VL chain combinations. [Table 3]

[0130] In addition to binding to ABCG2, the bispecific antibodies also bind to the TAAs listed in the table. The HCDR for anti-G2 antibodies can be as shown in Table 2. The second type of bispecific antibody The sequence in the VH region is shown below. Trastuzumab heavy chain: The VH region of JPEG2026053316000037.jpg51161 is underlined. CDR follows Kabat naming conventions and is shown in bold. It is being done.

[0131] Necitumumab heavy chain: The JPEG2026053316000038.jpg 55163VH region is underlined. HCDR1-3, following Kabat naming conventions, are shown in bold. It is being done.

[0132] Atezolizumab heavy chain: The JPEG2026053316000039.jpg 53163VH region is underlined. HCDR1-3, following Kabat naming conventions, are shown in bold. It is being done.

[0133] 5F9 VH chain: JPEG2026053316000040.jpg23163 HCDR1-3, which conform to Kabat, are shown in bold and underlined.

[0134] In some embodiments, bispecific antibodies that bind to both G2 and TAA are shown in Table 2. First VH including HCDR1-3 from the enumerated anti-G2 antibodies (e.g., G2.255) The second chain contains HCDR1-3 from an anti-TAA antibody (e.g., anti-CD47 antibody 5F9) It may contain a VH chain and a common VL chain, the VL chain being an anti-MDR1 antibody (e.g., MRK16). Includes LCDR1-3 from there.

[0135] In some embodiments, bispecific antibodies that bind to both G2 and TAA are shown in Table 2. HCDR1-3 from the listed anti-G2 antibodies, LCD from the anti-G2 antibodies listed in Table 2 Including R1-3, HCDR1-3 and LCDR1-3 are the same as those listed in Table 2. The first VL chain may be an antibody or the VH and VL chains of two different antibodies, and HCDR1-3 from anti-TAA antibodies such as anti-TAA antibodies described in the detailed document and specified herein The anti-TAA antibodies described include LCDR1-3 from anti-TAA antibodies, and HCDR1- 3 and LCDR1-3 are either from the same anti-TAA antibody or from two different antibodies. The first VH chain and the first VL chain are bound to G2, and the second VH chain is also included. The VH chain and the second VL chain bind to TAA.

[0136] In some embodiments, the target antibody is a recombinant or modified antibody, for example, a chimeric antibody. These are humanized, deimmunized, or in vitro-generated antibodies. When used herein, The terms “recombinant” or “modified” antibodies mean (i) transfected into host cells (ii) Recombinant combinatorial antibodies expressed using a recombinant expression vector (iii) Antibodies isolated from the antibody library, (iii) Gene induction in the human immunoglobulin gene Antibodies isolated from the animals (e.g., mice), or (iv) human immunoglobulins. Prepared by any other means involving splicing of a gene sequence to another DNA sequence, Antibodies, etc., that are currently produced, generated, or isolated, prepared, expressed, produced, or otherwise prepared by recombinant means. It is intended to include all antibodies to be isolated. Such recombinant antibodies may be humanized, CDR This includes grafted, chimeric, deimmunized, and in vivo-generated antibodies, which can be selected as needed. It may contain a constant region derived from a human germline immunoglobulin sequence.

[0137] Modified antibodies are those in which any antibody domain may be altered from its naturally occurring form. It may include, or may include, a modified domain. In some embodiments, the modified antibody is , modified Fc domains including modified CH2 and / or modified CH3 domains It may include modified heavy chains containing n. In some cases, modified Fc domains may be included. For example, but not limited to, disclosures incorporated in their entirety by reference herein Gunasekeran et al, (2010) Journal of Biol This is described in *Ogical Chemistry* 285, 19637-19646. Through the use of the procedure, the electrostatic steering effect can be utilized. In some cases, two Heavily specific antibodies are assembled by charge pair substitution in the CH3 domain, for example, one of them The heavy chain was modified to include the K392D and K409D substitutions (referred to as "DD"), The other heavy chain is modified to include the E356K and D399K substitutions (referred to as "KK"). This includes, but is not limited to, cases where charge pairs are substituted. Merges may preferentially form. The numbering of amino acid substitutions is based on the EU numbering system of Ig HC. Follow Tem.

[0138] In some cases, the antibodies of this disclosure involve charge pair substitution. Therefore, the antibodies of this disclosure do not involve charge pair substitution. In some cases, preference for the desired chain Alternative means to promote heterodimer formation may be used.

[0139] In some cases, the modified heavy chain was knob-in-to-hole. May include (to-hole) modifications. "No-into-hole" amino acid modifications are used in antibody engineering. This is a rational design strategy for producing multispecific antibodies, including bispecific IgG antibodies. In this context, it is used for the heterodimerization of heavy chains. For example, in the Nobu Into Hole battle The abbreviation is incorporated into a bispecific antibody produced from two monoclonal antibodies with different specificities. At that time, the amino acid changes were manipulated to add "NO" to the CH3 of the heavy chain of monoclonal antibody 1 (mAb1). A hole is created in the CH3 of the heavy chain of monoclonal antibody 2 (mAb2). Knobs can be represented, for example, by large amino acids such as tyrosine (Y), but on the other hand, Holes can be represented by small amino acids such as threonine (T). For example, knobs The in-hole modification involves the substitution of the first CH3 domain with T22Y and its partner C A Y86T substitution can be created in the H3 domain. An example of a knob-in-to-hole modification is shown in Cart. er, J. Immunol. Methods, 248(1-2):7-15(2001) , Ridgway, JBet al.Protein Eng.9(7):617- 2 (1996), and Merchant, A Met al. Nat. Biotech These disclosures are described in nol.16(7):677-81(1998), and their disclosures are in this The entirety of these will be incorporated into the specification. A pair formed knob-into-hole modified domain or In antibodies produced, bispecific heterodimers generally show the dominant proportion. The substitution numbering follows the EU numbering system in Ig HC.

[0140] As mentioned above, the target anti-ABCG2 antibody is specific to one or more epitopes of ABCG2. It binds to the target. Therefore, the epitope is the ABCG2 epitope. Anti-ABCG2 The size of the ABCG2 epitope bound by the antibody can change, and the ABCG2 epitope For example, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa , 12aa, 4aa~10aa, 5aa~10aa, 6aa~10aa, 4aa~8aa This includes but is not limited to 5aa~8aa, 6aa~8aa, etc., and 3aa or less~1 Formed by polypeptides having continuous extensions of the ABCG2 sequence, which can be in the range of 2aa or more. This includes cases where it is done.

[0141] In some embodiments, the ABCG2 epitope is, for example, a human ABCG2 sequence. : MSSSNVEVFIPVSQGNTNGFPATASNDLKAFTEGAVLS FHNICYRVKLKSGFLPCRKPVE KEILSNINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPS GLSGDVLINGAPRPANFKCN SGYVVQDDVVMGTLTVRENLQFSAALRLATTMTNHEKNER INRVIQELGLDKVADSKVGT QFIRGVSGGERKRTSIGMELITDPSILFLDEPTTGLDSST ANAVLLLLKRMSKQGRTIIF SIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFESA GYHCEAYNNPADFFLDIING DSTAVALNREEDFKATEIIEPSKQDKPLIEKLAEIYVNSS FYKETKAELHQLSGGEKKKK ITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQII VTVVLGlvIGAIYFGLKNDS TGIQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYI SGYYRVSSYFLGKLLSDLLP MRMLPSIIFTCIVYFMLGLKPKADAFFVMMFTLMMVAYSA SSMALAIAAGQSVVSVATLL MTICFVFMMIFSGLLVNLTTIASWLSWLQYFSIPRYGFTA LQHNEFLGQNFCPGLNATGN NPCNYATCTGEEYLVKQGIDLSPWGLWKNHVALACMIVIF LTIAYLKLLFLKKYS (Sequence ID 393), or its ECD1 (417~ 428): KNDSTGIQNRAG (Sequence ID 1), its ECD2 (499~506) :LKPKADAF (Sequence ID 2), its ECD3 (557-630): NLTTIA SWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATGNNP CNYATCTGEEYLVKQGIDLSPWGLWKNH (Sequence ID 3), or Mu s musculus ABCG2 sequence: MSSSNDHVLVPMSQRNNNGL PRTNSRAVRTLAEGDVLSFHHITYRVKVKSGFLVRKTVEK EILSDINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPKG LSGDVLINGAPQPAHFKCCS GYVVQDDVVMGTLTVRENLQFSAALRLPTTMKNHEKNERI NTIIKELGLEKVADSKVGTQ FIRGISGGERKRTSIGMELITDP SILFLDEPTTGLDSSTA NAVLLLLKRMSKQGRTIIFS IHQPRYSIFKLFDSLTLLASGKLVFHGPAQKALEYFASAG YHCEPYNNPADFFLDVINGD SSAVMLNREEQDNEANKTEEPSKGEKPVIENLSEFYINSA IYGETKAELDQLPGAQEKKG TSAFKEPVYVTSFCHQLRWIARRSFKNLLGNPQASVAQLI VTVILGLIIGAIYFDLKYDA AGMQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYI SGYYRVSSYFFGKVMSDLLP MRFLPSVIFTCVLYFMLGLKKTVDAFFIMMFTLIMVAYTA SSMALAIATGQSVVSVATLL MTIAFVFMMLFSGLLVNLRTIGPWLSWLQYFSIPRYGFTA LQYNEFLGQEFCPGFNVTDN STCVNSYAICTGNEYLINQGIELSPWGLWKNHVALACMII IFLTIAYLKLLFLKKYS (Sequence ID 394), or its ECD1 (41 5~428): DLKYDAAGMQNRAG (Sequence ID 395), its ECD2 (4 99~506): LKKTVDAF (Sequence ID 396), its ECD3 (557~632 ):NLRTIGPWLSWLQYFSIPRYGFTALQYNEFLGQEFCPG FNVTDNSTCVNSYAICTGNEYLINQGIELSPWGLWKNH(distribution Serial number 397), non-human primate sequence, e.g., Macaca fascicularis (Cynomolgus macaque) Sequence: MSSSNVEVFIPMSQENTNGFPTTTSNDR KAFTEGAVLSFHNICYRVKVKSGFLPGRKPVE KEILSNINGIMKPGLNAILGPTGGGKSSLLDVLAARKDPS GLSGDVLINGALRPTNFKCN SGYVVQDDVVMGTLTVRENLQFSAALRLPTTMTTNHEKNER INRVIQELGLDKVADSKVGT QFIRGVSGGERKRTSIGMELITDPSILFLDEPTTGLDSST ANAVLLLLKRMSKQGRTIIF SIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFESA GYHCEAYNNPADFFLDIING DSTAVALNREEDFKATEIIEPSKRDKPLVEKLAEIYVDSS FYKETKAELHQLSGGEKKKK ITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQII VTVILGLVIGAIYFGLNNDS TGIQNRAGVLFFLTTNQCFSSVSAVELFVVEKKLFIHEYI SGYYRVSSYFFGKLLSDLLP MRMLPSIIFTCIVYFMLGLKPTADAFFIMMFTLMMVAYSA SSMALAIAAGQSVVSVATLL MTICFVFMMIFSGLLVNLTTIASWLSWLQYFSIPRYGFTA LQHNEFLGQNFCPGLNATVN NTCNYATCTGEEYLTKQGIDLSPWGLWKNHVALACMIVIF LTIAYLKLLFLKKYS (Sequence ID 398), or its ECD1 (417~ 428):NNDSTGIQNRAG (Sequence ID 399), its ECD2 (499~50 6): LKPTADAF (SEQ ID NO: 400), its ECD3 (557-630): NL TTIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNAT VNNTCNYATCTGEEYLTKQGIDLSPWGLWKNH(Sequence ID 401) ), or Pan troglodytes ABCG2 sequence:MSSSNVEVFIPM SQGNTNGFPATTSNDLKAFTEGAVLSFHNICYRVKLKSGF LPCRKPVEKEILSNINGIMKPGLNAILGPTGGGKSSLLDV LAARKDPSGGLSGDVLINGAPRPANFKCNSGYVVQDDVVMG TLTVRENLQFSAALRLPTTMTNHEKNERINRVIQELGLDK VADSKVGTQFIRGVSGGERKRTSIGMELITDPSILFLDEP TTGLDSSTANAVLLLLKRMSKQGRTIIFSIHQPRYSIFKL FDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAYNNPAD FFLDIINGDSTAVALNREEDFKATEIIEPSKQDKPLIEKL AEIYVNSSFYKETKAELHQLSGGEKKKKITVFKEISYTTS FCHQLRWVSKRSFKNLLGNPQASIAQIIVTVILGLVIGAI YFGLKNDSTGIQNRAGVLFFLTTNQCFSSVSAVELFVVEK KLFIHEYISGYYRVSSYFLGKLLSDLLPMRMLPSIIFTCI VYFMLGLKPKADAFFVMMFTLMMVAYSASSMALAIAAGQS VVSVATLLMTICFVFMMIFSGLLVNLTTIASWLSWLQYFS IPRYGFTALQHNEFLGQNFCPGLNATGNNPCNYATCTGEE YLVKQGIDLSPWGLWKNHVALACMIVIFLTIAYLKLLFLK ABCG2 includes, but is not limited to, KYS (SEQIDNO:402). Continuous elongation of at least approximately 75%, at least approximately 80%, at least approximately 85%, less Both approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 1 It can be formed by polypeptides having 00% amino acid sequence identity.

[0142] In some embodiments, the ABCG2 epitope is a mutated ABCG2 polypeptide It can be formed by cytoplasm. Mutant ABCG2 polypeptide is human ABCG It may be derived from two polypeptides. The human ABCG2 polypeptide has an open three-dimensional structure. This may include mutations that result in the ABCG2 polypeptide. It is a human mutant with an open three-dimensional structure. The ABCG2 polypeptide sequence provided herein is the human ABCG2 polypeptide sequence. Referenced and numbered, may include substitution:E211Q. In certain embodiments, open standing The mutant human ABCG2 polypeptide with a body structure has the amino acid sequence: MSSSNVEV FIPVSQGNTNGFPATASNDLKAFTEGAVLSFHNICYRVKL KSGFLPCRKPVEKEILSNINGIMKPGLNAILGPTGGGKSS LLDVLAARKDPSGGLSGDVLINGAPRPANFKCNSGYVVQDD VVMGTLTVRENLQFSAALRLATTMTNHEKNERINRVIQEL GLDKVADSKVGTQFIRGVSGGERKRTSIGMELITDPSILF LDQPTTGLDSSTANAVLLLLKRMSKQGRTIIFSIHQPRYS IFKLFDSLTLLASGRLMFHGPAQEALGYFESAGYHCEAYN NPADFFLDIINGDSTAVALNREEDFKATEIIEPSKQDKPL IEKLAEIYVNSSFYKETKAELHQLSGGEKKKKITVFKEIS YTTSFCHQLRWVSKRSFKNLLGNPQASIAQIIVTVVLGLV IGAIYFGLKNDSTGIQNRAGVLFFLTTNQCFSSVSAVELF VVEKKLFIHEYISGYYRVSSYFLGKLLSDLLPMRMLPSII FTCIVYFMLGLKPKADAFFVMMFTLMMVAYSASSMALAIA AGQSVVSVATLLMTICFVFMMIFSGLLVNLTTIASWLSWL QYFSIPRYGFTALQHNEFLGQNFCPGLNATGNNPCNYATC TGEEYLVKQGIDLSPWGLWKNHVALACMIVIFLTIAYLKL At least 80% (for example, at least 85%) of LFLKKYS (SEQ ID NO: 403) %, at least about 90%, at least about 95%, at least about 98%, at least about 99% It may contain amino acid sequences that are identical in %, or 100%).

[0143] In some embodiments, the mutated ABCG2 polypeptide has a closed three-dimensional structure. Derived from human ABCG2 polypeptide containing a mutation that results in the ABCG2 polypeptide it possesses. It is possible. A mutant human ABCG2 polypeptide having a closed three-dimensional structure is provided herein. The sequence of the human ABCG2 polypeptide was referenced and numbered, substitution: K86M. S87A; K86M, S87A, Q126A; or K86M, S87A, Q126A, R May contain 246E. In certain embodiments, mutant human ABCG having a closed three-dimensional structure. Polypeptide 2 has the amino acid sequence: MSSSNVEVFIPVSQGNTNGFPATASNDLKAFTEGAVLSF HNICYRVKLKSGFLPCRKPVEKEILSNINGIMKPGLNAIL GPTGGGMASLLDVLAARKDPSGGLSGDVLINGAPRPANFKC NSGYVVQDDVVMGTLTVRENLQFSAALRLATTMTNHEKNE RINRVIQELGLDKVADSKVGTQFIRGVSGGERKRTSIGME LITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTII FSIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFES AGYHCEAYNNPADFFLDIINGDSTAVALNREEDFKATEII EPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEKKK KITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQI IVTVVLGLVIGAIYFGLKNDSTGIQNRAGVLFFLTTNQCF SSVSAVELFVVEKKLFIHEYISGYYRVSSYFLGKLLSDLL PMRMLPSIIFTCIVYFMLGLKPKADAFFVMMFTLMMVAYS ASSMALAIAAGQSVVSVATLLMTICFVFMMIFSGLLVNLT TIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATG NNPCNYATCTGEEYLVKQGIDLSPWGLWKNHVALACMIVI FLTIAYLKLLFLKKYS (Sequence ID 404), MSSSNVEVFIPVSQGNTNGFPATASNDLKAFTEGAVLSF HNICYRVKLKSGFLPCRKPVEKEILSNINGIMKPGLNAIL GPTGGGMASLLDVLAARKDPSGGLSGDVLINGAPRPANFKC NSGYVVADDDVVMGTLTVRENLQFSAALRLATTMTNHEKNE RINRVIQELGLDKVADSKVGTQFIRGVSGGERKRTSIGME LITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTII FSIHQPRYSIFKLFDSLTLLASGRLMFHGPAQEALGYFES AGYHCEAYNNPADFFLDIINGDSTAVALNREEDFKATEII EPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEKKK KITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQI IVTVVLGLVIGAIYFGLKNDSTGIQNRAGVLFFLTTNQCF SSVSAVELFVVEKKLFIHEYISGYYRVSSYFLGKLLSDLL PMRMLPSIIFTCIVYFMLGLKPKADAFFVMMFTLMMVAYS ASSMALAIAAGQSVVSVATLLMTICFVFMMIFSGLLVNLT TIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATG NNPCNYATCTGEEYLVKQGIDLSPWGLWKNHVALACMIVI FLTIAYLKLLFLKKYS (Sequence ID 405), or MSSSNVEVFIPVSQGNTNGFPATASNDLKAFTEGAVLSF HNICYRVKLKSGFLPCRKPVEKEILSNINGIMKPGLNAIL GPTGGGMASLLDVLAARKDPSGGLSGDVLINGAPRPANFKC NSGYVVADDDVVMGTLTVRENLQFSAALRLATTMTNHEKNE RINRVIQELGLDKVADSKVGTQFIRGVSGGERKRTSIGME LITDPSILFLDEPTTGLDSSTANAVLLLLKRMSKQGRTII FSIHQPEYSIFKLFDSLTLLASGRLMFHGPAQEALGYFES AGYHCEAYNNPADFFLDIINGDSTAVALNREEDFKATEII EPSKQDKPLIEKLAEIYVNSSFYKETKAELHQLSGGEKKK KITVFKEISYTTSFCHQLRWVSKRSFKNLLGNPQASIAQI IVTVVLGLVIGAIYFGLKNDSTGIQNRAGVLFFLTTNQCF SSVSAVELFVVEKKLFIHEYISGYYRVSSYFLGKLLSDLL PMRMLPSIIFTCIVYFMLGLKPKADAFFVMMFTLMMVAYS ASSMALAIAAGQSVVSVATLLMTICFVFMMIFSGLLVNLT TIASWLSWLQYFSIPRYGFTALQHNEFLGQNFCPGLNATG NNPCNYATCTGEEYLVKQGIDLSPWGLWKNHVALACMIVI At least 80% of FLTIAYLKLLFLKKYS (Sequence ID 406) (for example) then at least 85%, at least about 90%, at least about 95%, at least about 98% It may contain amino acid sequences that are identical by at least approximately 99%, or 100%.

[0144] The target anti-ABCG2 antibody exhibits high affinity binding to ABCG2. For example, the target anti-A BCG2 antibody is at least approximately 10 times stronger than human ABCG2. -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, young Or at least about 10 -12 M, or 10 -12 It binds with affinity greater than M. Target anti-AB The CG2 antibody targets the epitope present in ABCG2, approximately 10 times -7 M~about 10 -8 M, approx. 1 0 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10M~about 10 -1 1 M, or about 10 -11 M~about 10 -12 M, or 10 -12 Binds with affinity greater than M ru.

[0145] The target anti-ABCG2 antibody is related to other proteins with different sequences (for example, related but Epitopes formed by amino acids within different sequences (such as EP) are virtually bound. Not shown. Epitone is formed by amino acids in proteins that are related but have different sequences. The binding of the target anti-ABCG2 antibody to the epitope in ABCG2 is generally due to the binding of the target anti-ABCG2 antibody to the epitope in ABCG2. This is a nonspecific binding with substantially lower affinity than the specific binding of the anti-ABCG2 antibody. Qualitatively low affinity generally corresponds to at least 2 times, 3 times, 5 times, 10 times, 50 times, or 100 times higher affinity. The affinity is 500 times, or even 1000 times lower.

[0146] The target anti-ABCG2 antibody acts via the ABCG2 transporter, for example, human ABCG2. The transport of the target molecule can be reduced. For example, the target anti-ABCG2 antibody reduces transport, Compared to the degree of transport in the absence of ABCG2 antibody, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about It can be reduced by 70%, at least about 80%, at least about 90%, or more. .

[0147] In some embodiments, the target antibody includes an FR region which is a mammalian sequence, and it For example, rodents, non-human primates, and human sequences (for example, their respective heavy-chain FR coding sequences) Includes (coded by column).

[0148] The target antibodies were analyzed using 85 sequences in the VH region of the VH-VL pair of the antibodies shown in Table 2. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 Identity of %, 96%, 97%, 98%, 99%, or higher (including 100%). It may contain a heavy chain variable (VH) region that includes an amino acid sequence. The target antibodies are shown in Table 2. The sequence of the VL region of the VH-VL pair of the antibody was 85%, 86%, 87%, and 88%. %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Light chains containing amino acid sequences with %, 99%, or more (including 100%) identity are available. It can include variable (VL) regions.

[0149] The region and / or chain of the target antibody may be linked by one or more linker regions. However, it is not necessary. If present, the linker region is approximately 5 amino acids long. 0 amino acids, for example, with lengths of approximately 5aa to 10aa, approximately 10aa to 15aa, approximately 1 5aa ~ approx. 20aa, approx. 20aa ~ approx. 25aa, approx. 25aa ~ approx. 30aa, approx. 30aa ~ Approximately 35aa, approximately 35aa to approximately 40aa, approximately 40aa to approximately 45aa, or approximately 45aa to approximately 5 It can be 0aa.

[0150] A suitable linker for use with the target antibody is the "flexible linker." In this case, linker molecules generally allow a certain degree of flexible movement between linked regions. It is long enough to be effective. Linker molecules are generally about 6 to 50 atoms long. Linker molecules include, for example, arylacetylenes and ethylenes containing 2 to 10 monomer units. Glycol oligomers, diamines, diacids, amino acids, or combinations thereof It is possible that other linker molecules capable of binding to polypeptides may exist from the perspective of this disclosure. It can be used from.

[0151] A suitable linker can be easily selected, consisting of one amino acid (e.g., Gly) ~ 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids No acids, for example, 4 to 10 amino acids, 5 to 9 amino acids, Different lengths such as 6 to 8 amino acids, or 7 to 8 amino acids. It can be any of the following, and has 1, 2, 3, 4, 5, 6, or 7 mesh It could be an acid.

[0152] An example of a flexible linker is a glycine polymer (G) n , Glycine-Serimpo Rimmer (for example, (GS)) n GSGGS n (Sequence ID 407), and GGGS n (array (including the number 408, where n is at least an integer of 1), glycine-alanine polymer, Alanine-serine polymers and other flexible linkers known in the art It contains glycine and glycine-serine polymers, which are comparable in that both of these amino acids are It is unstructured and therefore can function as a neutral tether between components, This is of interest. Glycine polymers are formed when glycine is placed in the pip-psi space with alanine. It is accessed significantly more often than residues with longer side chains and is far less restricted. Therefore, it is of particular interest (Scheraga, Rev. Computation See al. Chem. 11173-142 (1992). For example, flexi Bulllinker includes GGSG (SEQ ID NO: 409), GGSGG (SEQ ID NO: 410), GS GSG (SEQ ID NO: 411), GSGGG (SEQ ID NO: 412), GGGSG (SEQ ID NO: 412) 3) This includes, but is not limited to, GSSSG (Sequence ID 414), etc. The design of the peptide conjugated to any of the above elements is, in whole or in part, f It can include a flexible linker, thereby allowing the linker to be flexible. It may include one or more parts that provide a car and a lower flexible structure. They will recognize this.

[0153] In other cases, the flexibility of the hinge region of the antibody of this disclosure is due to amino acid C22 Mutate 0 with serine or any other natural amino acid, remove C220, or the whole hin Either remove the hinge or replace the IgG1 hinge with an IgG3 hinge. It can be reduced. The light chains can be linked via their C-terminal cysteine ​​to form antibodies. This is similar to the situation observed in human isotype IgA2m. This is relative to Fc. This results in reduced Fab flexibility and consequently reduced crosslinking ability. Another strategy to reduce the flexibility of the IgG1 molecule is to use the IgG2 hinge to move the IgG1 hinge. This involves replacing it with a hinge or an IgG2-like hinge. Alternatively, a hinge similar to the IgG2 hinge is used. A variant of the IgG1 hinge can be introduced. This variant (TH7Δ6~9) This includes the mutation T223C and two deletions (K222 and T225), and additional cysteine This produces a short hinge having [a certain characteristic].

[0154] The substitution of mouse CDRs with the human variable domain framework ensures their correct spatial orientation. This can lead to the preservation of, for example, the Human Mutable Domain Framework, where CDR is It incorporates the same or similar three-dimensional structure as the resulting mouse variable domain framework. This is because the framework array is derived from the mouse variable frame of CDR. To obtain human variable domains from human antibodies that show high sequence identity with the variable domain. Therefore, this can be achieved. The heavy and light chain variable framework regions are the same or different. It can be derived from a human antibody sequence. The human antibody sequence is derived from a sequence of naturally occurring human antibodies. It can be a sequence, or it can be the consensus sequence of several human antibodies. Ru. Kettleborough et al., Protein Engineeri ng 4:773 (1991), Kolbinger et al., Protein See Engineering 6:971 (1993).

[0155] Determining the complementarity of mouse donor immunoglobulins and appropriate human acceptor immunoglobulins. Once the region is identified, the next step is to optimize the characteristics of the resulting humanized antibody. The task is to determine which residues should be substituted if we were to replace any residues from these components. Generally, the introduction of mouse residues enhances the human-anti-mouse antibody (HAMA) response in humans. To increase the risk of antibody induction, mouse amino acid residues are used to replace human amino acid residues. Substitutions should be kept to a minimum. Approved in the field for determining the immune response. This method allows monitoring of HAMA responses in specific patients or during clinical trials. Yes. Patients who have been administered humanized antibodies will be immune at the start of the therapy and throughout the entire course of the treatment. Genetic evaluation can be performed. HAMA response can be measured using, for example, surface plasmon resonance (BI) technology. Using methods known to those skilled in the art, including ACORE and / or solid-phase ELISA analysis, the patient It is measured by detecting antibodies against humanized therapeutic reagents in serum samples from individuals. In many embodiments, the target humanized antibody substantially evokes the HAMA response in human subjects. It does not trigger.

[0156] Based on the CDR three-dimensional structure and / or the potential effects of these on antigen binding, A specific amino acid from the variable region framework residue is selected for substitution. (Mouse) The unnatural juxtaposition of the CDR region and the human variable framework region may result in structural suppression. This results in a decrease in binding affinity unless corrected by substitution of specific amino acid residues. Rasu.

[0157] The selection of amino acid residues for substitution is determined in part by computer modeling. It is possible to generate three-dimensional images of immunoglobulin molecules using a computer tool. The hardware and software are known in the art. Generally, molecular models It is generated starting from the structure of the immunoglobulin chain or its domain that has been elucidated. The modeled chain is based on its amino acid sequence similarity to the elucidated three-dimensional structure of the chain or domain. The chain or domain that shows the greatest sequence similarity after comparison is used as the starting point for constructing the molecular model. Selected as follows: A strand or domain that shares at least 50% sequence identity is selected as the model. Selected for the purpose of sizing, preferably an array of at least 60%, 70%, 80%, and 90% Those that share identity or more are selected for modeling. The starting point is revealed. The structure is modeled after the actual amino acids within the immunoglobulin chain or domain, and the starting structure. It is modified to allow for differences between the amino acids within it. The modified structure is then multiplied. It is assembled into a composite immunoglobulin. Finally, the model is minimized by energy. Furthermore, all atoms are within an appropriate distance from each other, and the bond length and angle are chemically acceptable. It becomes refined by verifying that it is within certain limits.

[0158] In some embodiments, the target antibody contains scFv polymers. For example, several In this embodiment, the target antibody is an scFv dimer (for example, two tandem scFv( scFv2) (including), scFv trimer (e.g., three tandem scFv(scFv3) ) including, scFv tetramer (e.g., including four tandem scFv(scFv4)) It is either a single scFv or a polymer of more than four scFvs (e.g., in tandem). Monomers are linkers with a length of approximately 2 to 15 amino acids, for example, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11a It can be connected via linkers a, 12aa, 13aa, 14aa, or 15aa. Suitable linkers include, for example, (Gly) x , (sequence number 420) is included, and x is The integers are between 2 and 15. Other suitable linkers are those discussed above. In the embodiment, each of the scFv monomers in the target scFV polymer is as described above. It is then humanized. In certain embodiments, bispecific antibodies are humanized in any molecular shape known in the literature. It may be a formula. For example, the bispecific antibody of this disclosure is described by Spiess C. et al. This is described in Mol Immunol. 2015 Oct;67(2 Pt A):95-106. It may have the molecular form shown.

[0159] In some embodiments, the target antibody is the constant region of the immunoglobulin (e.g., Fc Includes the region. The Fc region, if present, can be a human Fc region. Steady-state region If present, the antibody may contain both a light chain and a heavy chain constant region. Suitable heavy chain The steady-state region includes the CH1, hinge, CH2, CH3, and CH4 regions. The antibodies listed include all types of antibodies, including IgM, IgG, IgD, IgA, and IgE. The normal region, and any isotype including IgG1, IgG2, IgG3, and IgG4. It contains antibodies having the following characteristics. A suitable example of a heavy chain Fc region is human isotype IgG1 Fc. The light chain constant region can be lambda or kappa. The antibody of the present invention (for example, this The humanized antibody of the invention may contain sequences from two or more classes or isotypes. The antibody exists as a tetramer containing two light chains and two heavy chains, with the heavy chains and light chains being separate components. As Fab, Fab'F(ab')2, and Fv, or as the heavy chain and light chain variable domains It can be expressed as a single-chain antibody linked via a pacer.

[0160] In some embodiments, the target antibody has a free thiol (-SH) at its carboxyl terminus. ) containing a group, and using a free thiol group, the antibody is converted into a second polypeptide (e.g., the target antibody) It can be conjugated to other antibodies (including this one), scaffolds, carriers, etc.

[0161] The target antibodies are, for example, glutaraldehyde, homobifunctional crosslinking agents, or heterobifunctional Using a crosslinking agent, the second part (e.g., lipids, polypeptides other than the target antibody, synthetic polypropylene) is crosslinked. It can be covalently bonded to (milli, carbohydrates, toxins, etc.). Glutaraldehyde is a poly Lipeptides are crosslinked via their amino moieties. Homobifunctional crosslinking agents (e.g., Homodifunctional imide esters, homodifunctional N-hydroxysuccinimidyl (NHS) Esters, or homobifunctional sulfhydryl reactive crosslinking agents, are two or more identical reactive agents. A crosslinking agent is added to a solution containing a mixture of polypeptides that include portions to be linked. It can be used in a one-step reaction procedure. Homodifunctional NHS esters and imides. Esters crosslink amine-containing polypeptides. At a mildly alkaline pH, Midoesters react only with primary amines to form imidamides, and cross-linked polypeptides The total charge of the cydo is unaffected. Homo-bifunctional sulfhydryl reactive crosslinking agents include bisma. Reimidohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFD NB), and 1,4-bis[3-(2-pyridyldithio)propionamide]butane (D It includes PDPB.

[0162] Compositions and Formulations This disclosure provides a composition comprising a target antibody. The target antibody composition comprises, in addition to the target antibody, Salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as Tri s buffering agent, histidine buffering agent, N-(2-hydroxyethyl)piperazine-N'-(2- Ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES ), 2-(N-morpholino)sodium ethanesulfonate (MES), 3-(N-mol) Horino) Propanesulfonic acid (MOPS), N-Tris[hydroxymethyl]methyl-3 - Aminopropanesulfonic acid (TAPS), etc.; solubilizers; surfactants, for example, Twe Among nonionic surfactants such as EN-20; protease inhibitors; glycerol, etc. It can contain one or more of the following.

[0163] The compositions of this disclosure also include pharmaceutical compositions comprising antibodies described herein. Generally The formulation contains an effective amount of the target antibody. "Effective amount" refers to the amount of the desired result, for example, in the target. Sufficient to produce a reduction in cancer, a decrease in the cancer growth rate in the target population, and an improvement in cancer symptoms. This refers to a dosage. Generally, the desired outcome is at least the reduction of cancer symptoms compared to the control group. The reductions are in cancer growth and tumor size. The target antibody is It can be delivered or formulated by means of circumventing the blood-brain barrier.

[0164] In some cases, antibodies may contain delivery enhancers, and such enhancers may be present in the blood. If it can facilitate crossing of the fluid-brain barrier, if it can facilitate increased permeability (for example, efficient passage) This includes things like enabling skin delivery.

[0165] In some cases, the antibodies of this disclosure do not need to be administered in formulations having a delivery enhancer. Good. In some cases, the antibodies of this disclosure themselves cross the blood-brain barrier. It may enhance permeability. In some cases, the antibodies of this disclosure are antitumor agents, for example, Used as a delivery enhancer to facilitate the crossing of the blood-brain barrier by immunotherapeutic agents or chemotherapeutic agents. In some cases, the antibody of this disclosure may be used with other antibodies or chemotherapeutic agents. Activators can cross the blood-brain barrier, the blood-cerebrospinal fluid (CSF) barrier, the blood-testis barrier, or blood- It can be used as a delivery accelerator to facilitate crossing of the placental barrier.

[0166] In the method described, the target antibody is an optional antibody capable of producing the desired therapeutic or diagnostic effect. It can be administered to the host using convenient means. Therefore, the drug can be used for treatment. It can be incorporated into various formulations for targeted administration. More specifically, the target antibody is appropriate Formulation into pharmaceutical compositions by combining with pharmaceutically acceptable carriers or diluents. It can be used in the form of tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and air It can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as rosols.

[0167] In formulations, the target antibody can be administered together with pharmaceutically acceptable excipients. or they, either alone or in appropriate combination with other pharmaceutically active compounds, They can be used in combination. The following methods and excipients are merely illustrative and not limiting. I can't.

[0168] The target antibodies are plant or other similar oils, synthetic aliphatic acid glycerides, high aliphatic acids, or It is soluble in aqueous or non-aqueous solvents such as propylene glycol esters, as needed. Along with conventional additives such as phosphates, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives, These can be formulated into an injectable preparation by dissolving, suspending, or emulsifying them. .

[0169] A pharmaceutical composition containing the target antibody is obtained by selectively and physiologically obtaining an antibody having the desired purity. Mix with acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonic agents. It is prepared by the following: Permitted carriers, excipients, and / or stabilizers are used. It is nontoxic to recipients in terms of dosage and concentration, and is not toxic to phosphoric acid, citrate, and other organic acids. Buffering agent; contains ascorbic acid, glutathione, cysteine, methionine, and citric acid. Antioxidants; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cre) Zole, p-chlor-m-cresol, methyl or propylparaben, benzal chloride Conium, or combinations thereof; arginine, glycine, ornithine, lysine, hi Stidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, pheny Lualanine, tyrosine, tryptophan, methionine, serine, proline, and the same Combinations of amino acids; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (approximately 10 residues) Polypeptides (less than); proteins such as gelatin or serum albumin; EDTA Which chelating agent; trehalose, sucrose, lactose, glucose, mannose, ma Fructose, galactose, fructose, sorbose, raffinose, glucosamine, N - Sugars such as methylglucosamine, galactosamine, and neuraminic acid; and / or T ween, Brij Pluronics, Triton-X, or polyethylene It contains nonionic surfactants such as PEG.

[0170] The pharmaceutical composition is in liquid form, lyophilized form, or liquid form reconstituted from the lyophilized form. The lyophilized preparation may be reconstituted with sterile solution before administration.

[0171] The typical antibody concentrations in the target pharmaceutical composition are approximately 1 mg / mL to approximately 200 mg / mL, or Approximately 50 mg / mL to approximately 200 mg / mL, or approximately 150 mg / mL to approximately 200 mg / mL It could be within the range.

[0172] Aqueous antibody formulations are prepared in a pH buffer solution at, for example, approximately 4.0 to 7.5, or approximately 5.0 to 7.5. It can be prepared at a pH in the range of approximately 6.0, or alternatively, approximately 5.5. Examples of suitable buffers include phosphate-, histidine-, citrate-, succinate-, and acetate-buffers. The formulation includes the agent and other organic acid buffers. The buffer concentration is, for example, the desired concentration of the buffer and formulation. Depending on the tension, it can be approximately 1 mm to 100 mm, or approximately 5 mm to 50 mm. .

[0173] In some embodiments, the aqueous formulation is isotonic, but hypertonic or hypotonic solutions are also available. It may be suitable. The term "isotonic" refers to the physiological salt solution or This indicates a solution that has the same tonicity as any other solution, such as serum. Isotonic solutions are approximately 5 mM to approximately It can be used in quantities of 350 mM, for example, in quantities between 100 mM and 350 nM.

[0174] Furthermore, surfactants are added to antibody preparations to reduce aggregation of the formulated antibodies, and / or to reduce aggregation of the antibodies. The formation of fine particles in the agent can be minimized, and / or adsorption can be reduced. Exemplary surfactants include Polyoxyethylene sorbitan fatty acid ester (Tween), polyoxyethylene Lukyl ether (Brij), alkylphenyl polyoxyethylene ether (Trit (on-X), polyoxyethylene-polyoxypropylene copolymer (Poloxame Contains r,Pluronic, and sodium dodecyl sulfate (SDS). Surface activity The exemplary concentration of the agent may range from approximately 0.001% to approximately 1% w / v.

[0175] Additionally, a freeze-protecting agent is added to freeze-dry unstable active ingredients (e.g., proteins). It can protect against destabilizing conditions during the process. For example, known cryoprotectants include sugars (glucosulfites). Polyols (including cereals and sucrose), mannitol, sorbitol, and glycerosol Contains (including ) and amino acids (including alanine, glycine, and glutamic acid). The freeze-protecting agent can be included in an amount of approximately 10 mM to 500 nM.

[0176] In some embodiments, the target formulation contains the target antibody and the drug identified above. Contains one or more of the following (e.g., surfactant, buffer, stabilizer, isotonic agent), ethanol, ba Dyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or one or more of the following: propylparaben, benzalkonium chloride, and combinations thereof. It is essentially free of preservatives. In other embodiments, the preservative is, for example, about 0.001 It is included in the formulation at a concentration in the range of approximately 2% (w / v).

[0177] For example, the target formulation can be a liquid or lyophilized formulation suitable for parenteral administration, approximately Target antibody at a concentration of 1 mg / mL to approximately 200 mg / mL, at least 1% at a concentration of approximately 0.001% to approximately 1%. One surfactant, approximately 1 mM to approximately 100 mM buffering agent, optionally approximately 10 mM to approximately 50 It can contain a 0 mM stabilizer and an isotonic agent of approximately 5 mM to approximately 305 mM, and approximately 4.0 to approximately It has a pH of 7.0.

[0178] The target antibody can be used in aerosol formulations administered by inhalation. The body is in pressurized, permissible propellants such as dichlorodifluoromethane, propane, and nitrogen. It can be formulated into a pharmaceutical product.

[0179] As used herein, the term “unit dosage form” refers to the subject matter of human and animal subjects. This refers to physically distinct units suitable as a unit dose, where each unit is pharmaceutically acceptable. The amount calculated in relation to the diluent, carrier, or vehicle is sufficient to produce the desired effect. It contains a predetermined amount of the compound of the present invention. The specifications of the target antibody are determined by the specific antibody used and the achievement The desired effect may depend on the pharmacodynamics associated with each antibody in the host.

[0180] The target antibody can be administered as an injectable formulation. Typically, an injectable set The product is prepared as a liquid solution or suspension and dissolved or suspended in a liquid vehicle before injection. Solid forms suitable for turbidity can also be prepared. The preparation may also be emulsified, or antibodies may be added to liposomes. It can be enclosed in a vehicle.

[0181] Suitable excipient vehicles include, for example, water, physiological saline, dextrose, glycerol, Ethanol, and combinations thereof. In addition, the vehicle may be used as needed. It may contain small amounts of auxiliary substances such as humectants, emulsifiers, or pH buffers. The actual method for preparing the state will be known or obvious to those skilled in the art.

[0182] Pharmacologically acceptable excipients such as vehicles, adjuvants, carriers, or diluents are generally It is readily available. Furthermore, pH adjusters, buffers, tonicity adjusters, stabilizers, wetting agents, etc. Any pharmaceutically acceptable adjuvants are generally readily available.

[0183] In some embodiments, the target antibody is formulated into a controlled-release formulation. Sustained-release preparation The material can be prepared using methods well known in the art.

[0184] Dosage The appropriate dosage should be determined by the attending physician or another qualified healthcare professional, based on various clinical factors. This can be decided. As is well known in the medical field, for any one patient The dosage depends on the patient's size, body surface area, age, the specific compound being administered, the patient's sex, and the duration of administration. , and many other factors including the route of administration, overall health status, and other drugs administered concurrently. It depends on the cause. The target antibody is administered at a dose of 1 ng / kg body weight to 20 mg / kg body weight, for example. 0.1 mg / kg body weight to 10 mg / kg body weight, for example, 0.5 mg / kg body weight to 5 mg / kg body weight. It may be administered in amounts equivalent to g body weight, but considering the factors mentioned above, it may be less than this exemplary range. Or a higher dose may be expected. If the regimen is administered by continuous infusion, 1 kilogram of body weight per minute. The amount can also be in the range of 1 μg to 10 mg per rum.

[0185] Those skilled in the art will know that the dose level depends on the specific antibody, the severity of the symptoms, and the subject's sensitivity to side effects. It will be easy to understand that this can change as a function of sex. The dosage can be easily determined by those skilled in the art through various means.

[0186] Route of administration The target antibody is a drug that can be administered via in vivo and ex vivo methods, as well as systemic and local routes of administration. It is administered to the individual using any available method and route suitable for physical delivery.

[0187] Conventional pharmacopoeia-acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, and topical administration. This includes intravenous, intra-arterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. The route of administration may be combined depending on the antibody and / or the desired effect, or as needed. The dosage may be adjusted accordingly. The target antibody composition can be administered in single or multiple doses. In some embodiments, the target antibody composition is administered orally. In its form, the target antibody composition is administered via the inhalation route. In some embodiments... In this configuration, the target antibody composition is administered intranasally. In some embodiments, the target antibody The composition is administered topically. In some embodiments, the target antibody composition is administered to the skull. It is administered intravenously. In some embodiments, the target antibody composition is administered intravenously.

[0188] The drug can be delivered via any available conventional drug delivery route, including systemic or topical routes. It can be administered to the host using conventional methods and routes. Generally, the present invention The intended routes of administration include enteral, parenteral, or inhalation routes, but not necessarily these. Not limited to this.

[0189] Other parenteral administration routes besides inhalation include topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, and spinal cord administration. This includes internal, intrasternal, and intravenous routes, i.e., any route of administration other than through the gastrointestinal tract. However, it is not limited to these. Parenteral administration involves systemic or local delivery of the target antibody. It can be done to deliver the drug. If systemic delivery is desired, the administration is typically carried out by the pharmaceutical company. This involves local or mucosal administration of the product, which may result in invasive or systemic absorption.

[0190] The target antibody can also be delivered to the target via enteral administration. The enteral administration route includes: Oral and rectal (e.g., using suppositories) delivery are included, but not necessarily limited to, these methods. do not have.

[0191] Treatment means improvement of symptoms related to at least the pathological condition affecting the host. The term "improvement" is used in a broad sense to refer to the improvement of cancer and / or cancer growth, as well as the pain associated with it. Parameters related to the pathological condition being treated, such as the severity of symptoms. Both refer to a decline. Therefore, treatment also addresses the pathological condition, or at least something related thereto. The symptoms are completely inhibited, for example, prevented from occurring, stopped, or terminated. As a result, the host is no longer in a pathological state, or at least characterized by a pathological state. This includes situations in which the person does not suffer from the symptoms.

[0192] Various subjects (the term "subject" is used herein to mean "individual" and "patient") (used interchangeably with the term) but treatable according to the method of this disclosure. Generally, such treatment Elephants are "mammals" or "mammals," and these terms refer to the order Carnivora (e.g., dogs and Cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., Widely used to describe organisms within the class Mammalia, including humans, chimpanzees, and monkeys. In some embodiments, the host is a human.

[0193] A kit containing a unit dose of the target antibody is provided, for example, in an oral dose or an injectable dose. In some embodiments, in addition to a container containing a unit dose, the pathological of the subject is... There is an informational document that explains the use of antibodies in the treatment of the condition and the associated benefits.

[0194] nucleic acid This disclosure provides nucleic acids comprising a nucleotide sequence encoding a target antibody. The encoded nucleotide sequence is intended for use in target cells (e.g., when synthesizing an encoded antibody). Expression of nucleotide sequences in cells that have been genetically modified to secrete and / or secrete One or more controllable elements, such as promoters and enhancers, that enable operation. It can be connected to.

[0195] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include lacI, lacZ, T3, This includes, but is not limited to, T7, gpt, lambda P, and trc. For expression in biological cells, suitable promoters include light chain and / or heavy chain immunoglobulins. Phosphorus gene promoter and enhancer element, cytomegalovirus immediate early stage Lomotor, herpes simplex virus thymidine kinase promoter, early and late SV4 0 promoter, promoter present in long-term terminal repeats from retroviruses, mouseme Tarothionein-I promoter, as well as various technically known tissue-specific promoters This includes, but is not limited to, those mentioned above.

[0196] The nucleotide sequence encoding the target antibody is used in the expression vector and / or cloning vector. It can be present in the target. If the target antibody contains two or more distinct polypeptides, 2 The nucleotide sequences encoding two polypeptides are crossed into the same or different vectors. It can be done. A separate polypeptide can be a separate promoter, one or more internal polypeptides. Ribosome entry site (IRES), one or more autocleavage sequences (e.g., P2A, T2A, Various strategies are used, such as E2A and 2A cleavage sequences (F2A, etc.), and combinations thereof. The expression can be expressed from a single nucleic acid or a single vector. The expression vector has selectable markers. - may include a replication origin, and other properties that provide replication and / or maintenance of the vector. Cut.

[0197] Numerous suitable vectors and promoters are known to those skilled in the art, and many of them are related to the target recombinant structure. These are commercially available for generating structures. The following vectors are provided as examples: Bacteria: pBs, PhageScript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagen e, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsal) a,Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1 , pSG(Stratagene)pSVK3, pBPV, pMSG, and pSVL(P (harmacia).

[0198] Expression vectors generally have convenient restriction sites located near the promoter sequence. This provides insertion of nucleic acid sequences encoding heterologous proteins. Selective operation in the expression host. Possible markers may exist. Suitable expression vectors include viral vectors (e.g., w Viral vectors based on cynia virus, poliovirus, adenovirus, adeno Associated viruses, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus Svectors (e.g., mouse leukemia virus, splenic necrosis virus, and Roussarcoma virus) S, Harvey's sarcoma virus, avid leukemia virus, human immunodeficiency virus, myeloproliferative meat This includes vectors derived from retroviruses such as tumor viruses and breast cancer tumor viruses. However, it is not limited to these.

[0199] For example, nucleic acids as described herein, in some cases, for example, cells are nucleic acids It can be introduced into cells by contact with it. Cells that have the introduced nucleic acid are Generally, these are referred to as genetically modified cells in this specification. Various nucleic acid delivery methods may be used. For example, naked nucleic acid delivery, viral delivery, chemical transfection, gene guns This includes, but is not limited to, these.

[0200] cell This disclosure relates to isolated genetically modified cells (e.g., in) that have been genetically modified with the target nucleic acid. The invention provides in vitro cells, ex vivo cells, cultured cells, etc. In some embodiments, The isolated target gene-modified cells can produce the target antibody. Several examples In this context, genetically modified cells can deliver antibodies to targets that require them, for example. Yes, it is possible. In some cases, genetically modified cells produce multispecific antibodies, screen It can be used for learning and / or discovery.

[0201] Suitable cells include eukaryotic cells such as mammalian cells, insect cells, and yeast cells, as well as bacterial cells. This includes prokaryotic cells such as [examples of prokaryotic cells]. The introduction of target nucleic acids into host cells is, for example, calcium phosphate. Umum precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection This can be achieved by sception, electroporation, or other known methods. .

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

[0203] In some cases, useful mammalian cells are derived from mammalian tissues or organs. It may include. In some cases, the cells used are kidney cells, for example, HEK Includes kidney cells from established kidney cell lines such as 293T cells.

[0204] In some cases, the cells of this disclosure may be immune cells. In this case, the term "immune cells" generally refers to hematopoietic stem cells (HSCs) produced in the bone marrow. Includes white blood cells (leukocytes) from which it originates. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells, and bone marrow-derived cells (neutrophils, eosinophils, basophils) This includes monocytes, macrophages, and dendritic cells. "T cells" include T helper cells (C D4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and cancer Includes all types of immune cells that express CD3, including M-delta T cells. "Harmful cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils. These cells can mediate cytotoxic responses.

[0205] In several cases, useful cells expressing antibodies such as the multispecific antibodies of this disclosure This may include producer T cells. It may also include nucleic acid sequences encoding the antibodies of this disclosure. In some cases, the manipulated producer T cells produced antibodies that were needed. It can be used to deliver something to a target.

[0206] In several cases, the immune cells of this disclosure have an ABCG2 binding domain, transmembrane domain Immunotherapy includes an immunoassay receptor (CAR) containing an intracellular signaling domain. Fector cells are included, and the ABCG2 binding domain is the variable heavy chain of the antibodies listed in Table 2. The pair of heavy chain complementarity determination regions (HCDR) and light chain C between the VH region and the variable light chain (VL) region. Includes DR (LCDR). In one embodiment, the intracellular signaling domain is at least One co-stimulatory molecule, e.g., 4-1BB (i.e., CD137), CD27, and / or It may contain one or more functional signaling domains derived from CD28. Intracellular signaling The signaling domain is a functional signaling domain derived from the co-stimulator molecule and the stimulator molecule It may include functional signaling domains.

[0207] Immune effector cells can be T cells. Immune effector cells are autologous cells. It is possible.

[0208] method As summarized above, the method of the present disclosure is a method of contacting cells with the antibody of the present disclosure. A method of treating a subject by a method involving the administration of the indicated antibody to the subject, as described in this application. Elements, such as antibodies, multispecific antibodies, compositions and formulations, nucleic acids, expression vectors, cells, etc. This includes a method for producing it.

[0209] As described above, the method of the present disclosure involves contacting cancer cells with the antibody of the present disclosure, for example, To detect the presence of ABCG2 expression in cancer cells, or to measure the expression level of ABCG2 in cancer cells. This includes measuring or promoting and / or enhancing the killing of cancer cells. In this case, the killing of cancer cells is due to the immune response that acts on cancer cells bound by antibodies. It is mediated by the response or immune cells. In some cases, the killing of cancer cells is, for example Therefore, as a result of antibody-mediated inhibition of ABCG2, the inhibition of cancer cell efflux is mediated. In some cases, killing cancer cells inhibits the cell elimination of cancer cells and the immune response. It is mediated in combination with a mediating response (e.g., via the Fc region of an antibody). In some cases, the cells that come into contact with the multispecific antibodies may be multidrug-resistant cancer cells. A method involving contacting cancer cells with the antibodies of this disclosure is used to chemically treat cancer cells, for example. Contact with additional therapies or activators, including therapeutic agents, immunotherapies, and radiotherapy agents. It may or may not include.

[0210] Contacting cancer cells with the multispecific antibodies of this disclosure generally involves, for example, multispecificity Compared to the level of cancer cell killing in the absence of antibodies, it enhances the killing of cancer cells. In a few cases, when additional activators are used, the enhanced killing of cancer cells is further... This can be shown in comparison to the lethality levels observed when the additional activator is used alone. Multiple specificity antibodies The amount of enhancement in cancer cell killing due to the body changes, from at least a 5% increase in cancer cell killing. It could be in the range of at least 90% or more, for example, at least 10%, at least 20%, At least 30%, at least 40%, at least 50%, at least 60%, and at least This includes, but is not limited to, 70%, or at least 80%. Such an increase is one This can be compared to contacting the additional activators mentioned above individually.

[0211] Enhanced killing of cancer cells can be evaluated by various means, for example, observational tests, Cell-based cytotoxic assays in vitro, flow cytometry, and cell viability assays. This includes, but is not limited to, methods of identification (e.g., using one or more cell viability stains). .

[0212] Treatment method This disclosure provides a method for treating cancer, which generally applies to individuals who require it. For example, an effective amount of the antibody provided herein is administered alone to an individual (for example, an individual with cancer). (as monotherapy) or in combination with one or more additional therapeutic agents (for example, in combination therapy) The administration of the antibodies in this disclosure is carried out by any convenient and appropriate delivery route. I can.

[0213] Therefore, administration may involve, for example, delivery of antibodies by injection, delivery of antibodies by infusion, and antibody Delivery of nucleic acids or expression vectors that represent a specific substance, administration to cells that express and secrete antibodies. Antibody delivery by means of ABCG2 binding domain, transmembrane domain, and intracellular signaling Immunoeffectors express chimeric antigen receptors (CARs) containing a signaling domain on the cell surface. - This includes the delivery of cells (e.g., CAR-T cells), and the ABCG2 binding domain is shown in Table 2. The list includes HCDRs and LCDRs of pairs of VH and VL regions, such as antibodies. The administration of nucleic acids that encode drugs, cells that express drugs, etc., involves contact with the drug, nucleic acids This may include contact with, or contact with, cells, etc.

[0214] In some embodiments, an effective amount of the target antibody is administered alone (e.g., as monotherapy) or Administer in one or more doses in combination with one or more additional therapeutic agents (e.g., in combination therapy). If so, compare the severity of cancer adverse effects to the severity of adverse effects in the absence of antibody treatment. And 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% This is an effective amount to reduce by %, or more.

[0215] In some embodiments, an effective amount of the target antibody is administered alone (e.g., as monotherapy) or Administer in one or more doses in combination with one or more additional therapeutic agents (e.g., in combination therapy). If so, it improves the cancer in the treated individual (i.e., slows the growth of the cancer). This stops cancer growth, reverses cancer growth, and kills cancer cells (including tumor cells, etc.) This is an effective amount to kill (or injure). For example, an effective amount of target antibody is sufficient for antibody-based treatment. Compared to the present, at least approximately 5%, at least approximately 10%, at least approximately 15%, and at least Approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 40%, and at least It reduces the cancer growth rate in individuals by approximately 50% or more, or in individuals It can reduce the size of the tumor.

[0216] In some cases, the subject may be treated systemically, with one or more additional target antibodies. This includes using with or without the reagent. "Systemic treatment" as used herein If applicable, this may include specific tumors (e.g., primary tumors or defined secondary tumors) or specific cancers. The target is the tissue containing the substance (for example, the liver in cases of liver cancer, the blood in cases of blood cancer, etc.). It means treatment that goes beyond just treating the body. Systemic treatment is generally directed towards the entire body of the subject. This includes, for example, whole-body radiotherapy, whole-body chemotherapy, whole-body immunotherapy, and combinations thereof. These may be the case, but are not limited to these.

[0217] In some cases, the subject may be treated locally, with one or more additional target antibodies. This includes using the reagent with or without it. "Local treatment" as used herein If so, it is directed specifically to the location of the tumor (e.g., the primary tumor or a defined secondary tumor). or tissue containing cancer (for example, the liver in cases of liver cancer, or the liver in cases of blood cancer) This refers to treatment specifically directed at the blood, etc. In some cases, Treatment also involves surrounding tissues such as tissue directly adjacent to the tumor, or tissues surrounding the tumor. It may be administered in a manner that affects the environment. Local treatment is generally used for tumors such as primary tumors. It does not affect or target tissues far from the cancer site, including the ulcer site. Useful topical therapies that can be administered in addition to the body or in combination with the target antibody include, for example, Surgery, local radiotherapy, local cryotherapy, local laser therapy, topical therapy, and combinations thereof. This includes, but is not limited to, combinations and other similar items.

[0218] In some embodiments, the target treatment method comprises a target antibody and one or more additional therapeutic agents. This involves administering [a specific agent]. Suitable additional therapeutic agents include chemotherapeutic agents, radiotherapy reagents, and immunotherapeutic agents. This includes, but is not limited to, reagents, other antibodies, or multispecific antibody preparations. Additional therapies that may be administered to the subject before, during, or after the administration of the indicated multispecific antibody are, for example, For example, the type of cancer, the patient's treatment history, their overall health, and / or any co-existing conditions. It will vary depending on many factors, including, for example, radiation therapy. This includes, but is not limited to, therapies such as denatured medicine, chemotherapy, and immunotherapy.

[0219] Radiation therapy involves radiation from an external source such as a beam, or from a small radiation source. This includes, but is not limited to, X-rays or gamma rays delivered by implantation.

[0220] Suitable antibodies for use in cancer treatment include naked antibodies, such as trastuzumab. Herceptin), bevacizumab (Avastin (trademark)), cetuximab (Er bitux(trademark), panitumumab (Vectibix(trademark)), ipilimumab (Y Ervoy (trademark), Rituximab (Rituxan), Alemtuzumab (Lemtr ada(trademark), ofatumumab (Arzerra(trademark)), olegobomab (Ova Rex (trademark), lambrolizumab (MK-3475), pertuzumab (Perjet a(trademark), ranibizumab (Lucentis(trademark), etc., and conjugated anti For example, gemtuzumab ozogamicin (Mylortarg(trademark), brentuxi Mabvedotin (Adcetris®), 90Y labeled ibritumomabuchiuxetane ( Zevalin (trademark), 131I-labeled Toshitsumoma (Bexxar (trademark)), etc. These are included, but not limited to.

[0221] Antibodies suitable for use in cancer treatment include antibodies produced against tumor-associated antigens. However, it is not limited to these. Such antigens include CD20, CD30, CD33, and CD 52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, Folate-binding proteins, gangliosides (e.g., GD2, GD3, GM2, etc.), Ley VEGF, VEGFR, Integrin Alpha-V-Beta-3, Integrin Alpha a-5-beta-1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPH A3, TRAILR1, TRAILR2, RANKL, FAP, Tenasyn, Program Death ligand 1 (PD-L1), androgen receptor (AR), Bruton's tyrosine kinase Ze(BTK), BCR-Abl, c-kit, PIK3CA, EML4-ALK, KRA S, ALK, ROS1, AKT1, BRAF, MEKJ, MEK2, NRAS, RAC1 This includes ESR1, CTLA-4, LAG-3, and TIM-3, but is not limited to these. Not specified. These antibodies are used in combination therapy with the anti-ABCG2 antibodies provided herein. or at least one antigen-binding moiety of these antibodies provided herein It can be administered as a multispecific antibody containing the antigen-binding moiety of an anti-ABCG2 antibody.

[0222] Traditional cancer therapies also include targeted therapies for cancer, such as HER2(ER) Ado-trastuzumab emtansine (Kadcyla) targets BB2 / neu (Approved for use in breast cancer), EGFR (HER1 / ERBB1), HER2 ( Afatinib (Gilotrif) targeting ERBB2 / neu (non-small cell lung cancer) Approved for use in (kidney) Alectinib (Al), which targets ALK, is approved for use in cell carcinoma and melanoma. Ecensa (approved for use in non-small cell lung cancer), a drug that targets CD52 Lemtuzumab (Campath) (approved for use in B-cell chronic lymphocytic leukemia) ), PD-L1-targeting atezolizumab (Tecentriq) (urothelial carcinoma, non Avelumab (Bav), which targets PD-L1, is approved for use in small cell lung cancer. encio) (approved for use in Merkel cell carcinoma), KIT, PDGFRβ, Axitinib (Inlyta) that targets VEGFR1 / 2 / 3 (used in renal cell carcinoma) (is approved), belimumab (Benlysta) (targets BAFF) Bellinostat (Bele), which targets HDACs, is approved for use in Todes. ODAQ (approved for use in peripheral T-cell lymphoma), targets VEGF ligands. Bevacizumab (Avastin) is used for cervical cancer, colorectal cancer, fallopian tube cancer, and glioblastoma. (Approved for use in non-small cell lung cancer, ovarian cancer, peritoneal cancer, and renal cell carcinoma), CD Blinatumomab (Blincyto), which targets 19 / CD3 (acute lymphoblastic leukemia) Borteso, which targets the proteasome (approved for use in disease-related pre-B cell use). MIB (Velcade) (Approved for use in multiple myeloma and mantle cell lymphoma) Bosulif, which targets ABL (in chronic myeloid leukemia), is used in chronic myeloid leukemia. (Approved), brentuximab vedotin (Adcetris) which targets CD30 (Approved for use in Hodgkin lymphoma and anaplastic large cell lymphoma), targets ALK The target drug is brigatinib (Alunbrig), which is approved for use in non-small cell lung cancer (ALK+). (Recognized), targeting FLT3, KIT, MET, RET, and VEGFR2, Caboza Ntinib (Cabometyx, Cometriq) (in myelin thyroid cancer, renal cell carcinoma) Carfilzomib (Kyprol), which targets the proteasome (approved for use), is) (approved for use in multiple myeloma), ALK-targeting ceritinib (Z ykadia) (approved for use in non-small cell lung cancer), EGFR (HER1 / E Cetuximab (Erbitux), which targets RBB1, is used for colorectal cancer and head and neck flat cancer. Cobimetinib (Cotell), which targets MEK, is approved for use in epithelial cancer. (Approved for use in melanoma), targeting ALK, MET, and ROS1 Rizotinib (Xalkori) (approved for use in non-small cell lung cancer), BRAF Dabrafenib (Tafinlar), which targets [a specific cell type], is used in melanoma and non-small cell lung cancer. (Approved), daratumumab (Darzalex), which targets CD38 (multiple bones) Dasatinib (Sprycel), which targets ABL (and is approved for use in myeloma) RANKL (approved for use in chronic myeloid leukemia and acute lymphoblastic leukemia) The targeted drug is denosumab (Xgeva) (approved for use in giant cell tumors of bone), B Dinutuximab (Unituxin), which targets 4GALNT1 (GD2) (pediatric neurology) Durvalumab (Imf), which targets PD-L1 (approved for use in blastoma) inzi) (approved for use in urothelial carcinoma), SLAMF7 (CS1 / CD3 Elotuzumab (Empliciti), which targets 19 / CRACC (in multiple myeloma) (Approved for use), enasidenib (Idhifa) which targets IDH2 (acute Approved for use in myeloid leukemia, it targets EGFR (HER1 / ERBB1) Erlotinib (Tarceva) is approved for use in non-small cell lung cancer and pancreatic cancer. (is present), everolimus (Afinitor) which targets mTOR (pancreas, gastrointestinal tract, or lung) Neuroendocrine tumors of origin, renal cell carcinoma, unresectable subependymal giant cell astrocytoma, and breast cancer. Gefitinib, which targets EGFR (HER1 / ERBB1) and is approved for use. (Iressa) (approved for use in non-small cell lung cancer), targets CD20. Ibritumomab tiuxetan (Zevalin) (approved for use in non-Hodgkin lymphoma) (is being used), ibrutinib (Imbruvica) which targets BTK (mantle cell regeneration) It is approved for use in lymphoma, chronic lymphocytic leukemia, and Waldenström's hypergammaglobulinemia. (Recognized), idelalisib (Zydelig) (targeting PI3Kδ) (chronic lymphopathy It has been approved for use in leukemia, follicular B-cell non-Hodgkin lymphoma, and small lymphocytic lymphoma. Imatinib (Gleevec) (G), which targets KIT, PDGFR, and ABL. It is approved for use in stromal tumors (Kit+), dermatofibrosarcoma protuberans, and multiple malignant hematological disorders. ipilimumab (Yervoy), which targets CTLA-4, is used in melanoma. (Approved), proteasome-targeting ixazomib (Ninlaro) (multiple Approved for use in genital myeloma), HER2 (ERBB2 / neu), EGFR (H Lapatinib (Tykerb) targets ER1 / ERBB1 (for breast cancer (HER2+)) Lenvatinib (Lenvim), which targets VEGFR2, is approved for use in ) a) Mid (approved for use in renal cell carcinoma and thyroid cancer), targeting FLT3 Staurin (Rydapt) (approved for use in acute myeloid leukemia (FLT3+)) ), necitumumab (Portrazz), which targets EGFR (HER1 / ERBB1) a) (Approved for use in squamous cell non-small cell lung cancer), HER2 (ERBB2 / n Neratinib (Nerlynx), which targets the EU (approved for use in breast cancer) Nilotinib (Tasigna), which targets ABL (approved for use in chronic myeloid leukemia). Niraparib (Zejula), which targets PARP (ovarian cancer, fallopian tube cancer) Nivolumab (Opdiv), which targets PD-1 (approved for use in peritoneal cancer), is approved for use in peritoneal cancer. o) (Colorectal cancer, head and neck squamous cell carcinoma, Hodgkin lymphoma, melanoma, non-small cell lung cancer) (Approved for use in renal cell carcinoma and urothelial carcinoma), CD20-targeting obinu Tuzumab (Gazyva) (approved for use in chronic lymphocytic leukemia and follicular lymphoma) (is present), ofatumumab ((Arzerra, HuMax-CD2) which targets CD20 0) Olapari (approved for use in chronic lymphocytic leukemia), a PARP-targeting drug Lynparza (approved for use in ovarian cancer) targets PDGFRα Oralatumab (Lartruvo) (approved for use in soft tissue sarcoma), E Osimertinib (Tagrisso), which targets GFR (not approved for use in non-small cell lung cancer) Palbociclib (Ibrance), which targets CDK4 and CDK6 (and is approved). Approved for use in breast cancer, EGFR (HER1 / ERBB1) targeting Nitumumab (Vectibix) (approved for use in colorectal cancer), HDAC Panobinostat (Farydak), which targets [the target of multiple myeloma], is approved for use in multiple myeloma. Pazopanib (Votrient) targets VEGFR, PDGFR, and KIT. (Approved for use in renal cell carcinoma), PD-1-targeting pembrolizumab ( Keytruda) (Classical Hodgkin lymphoma, melanoma, non-small cell lung cancer (PD-L1+) ), Approved for use in head and neck squamous cell carcinoma and solid tumors (MSI-H), HER Pertuzumab (Perjeta), which targets ERBB2 2 (H2) (for breast cancer (HE) Approved for use in R2+, ABL, FGFR1-3, FLT3, VEGFR Ponatinib (Iclusig), which targets 2 (chronic myeloid leukemia, acute lymphoblastic leukemia) Ramucirumab (Cyram), which targets VEGFR2, is approved for use in blood diseases. za) (In colorectal cancer, gastric cancer or esophagogastric junction (GEJ) adenocarcinoma, non-small cell lung cancer) (Approved for use), KIT, PDGFRβ, RAF, RET, VEGFR1 / 2 / Regorafenib (Stivarga) that targets 3 (colorectal cancer, gastrointestinal stromal tumor, liver Ribociclib, which targets CDK4 and CDK6 (approved for use in cell carcinoma) Kisqali) (Approved for use in breast cancer (HR+, HER2-)), CD2 Rituximab (Rituxan, Mabthera) targeting 0 (non-Hodgkin lymphocytes) It is approved for use in tumors, chronic lymphocytic leukemia, rheumatoid arthritis, and granulomatosis with polyangiitis. (is present), rituximab / hyaluronidase (Rituxan) which targets CD20 Hycela (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma) Romidepsin (Istodax), which targets HDACs (approved for use in tumors) (Approved for use in cutaneous T-cell lymphoma and peripheral T-cell lymphoma), PARP is targeted The target drug is rucaparib (Rubraca) (approved for use in ovarian cancer), JAK Ruxolitinib (Jakafi), which targets half of the myelofibrosis, is approved for use in myelofibrosis. (is present), siltuximab (Sylvant) which targets IL-6 (multicentric castle Approved for use in Mann disease, the target cyplucel-T (Provenge (Approved for use in prostate cancer), sonidecib (Odom) targets smoothing. zo) (approved for use in basal cell carcinoma), VEGFR, PDGFR, KIT, Sorafenib (Nexavar), which targets RAF (hepatocellular carcinoma, renal cell carcinoma, thyroid cancer) Temsirolimus (Torise), which targets mTOR (approved for use in cancer) l) (Approved for use in renal cell carcinoma), tositumomab (B) which targets CD20 exxar (approved for use in non-Hodgkin lymphoma), a MEK-targeting drug Lametinib (Mekinist) (approved for use in melanoma and non-small cell lung cancer) , trastuzumab (Herceptin) which targets HER2 (ERBB2 / neu) (Approved for use in breast cancer (HER2+) and gastric cancer (HER2+), EGFR Vandetanib (Capr) targets (HER1 / ERBB1), RET, and VEGFR2. Vemura (ELSA) (approved for use in myelin thyroid cancer), which targets BRAF. Fenib (Zelboraf) (approved for use in melanoma), targets BCL2 Venetoclax (Venclexta) is approved for use in chronic lymphocytic leukemia. (is present), PTCH, bismodegib (Erivedge) which targets smoothing (basal cells) (Approved for use in) Vorinostat (Zolinza), which targets HDAC (Approved for use in cutaneous T-cell lymphoma), targets PIGF and VEGFA / B. Ziv-aflibercept (Zaltrap) (approved for use in colorectal cancer) Examples include, but are not limited to, those described herein. These antibodies are provided herein. It may be administered as combination therapy with anti-ABCG2 antibodies.

[0223] Suitable biological response modifiers for use in connection with the methods disclosed herein include (1) tyrosine kinases (1) Inhibitors of RTK activity, (2) Inhibitors of serine / threonine kinase activity, (3) tumor (4) Apoptosis Receptor agonists, (5) interleukin-2, (6) interferon-α, (7) Interferon-γ, (8) colony-stimulating factors, (9) angiogenesis inhibitors, and (10 This includes, but is not limited to, tumor necrosis factor antagonists.

[0224] Chemotherapy agents or antitumor agents are non-peptide (i.e., non) that reduce the proliferation of cancer cells. These are protein-based compounds, including cytotoxic agents and cell proliferation inhibitors. (Chemotherapy agents) Non-limiting examples include alkylating agents (e.g., nitrosourea) and antimetabolites (e.g., melanin). Totrexate, antitumor antibiotics (e.g., anthracyclines), plant alkaloids (For example, vinca alkaloids, taxanes, etc.), toposiomerase inhibitors, and steroids It contains id hormones.

[0225] Drugs that act to reduce cell proliferation are known in the art and are widely used. These drugs include nitrogen mustard, nitrosourea, ethyleneimine derivatives, This includes, but is not limited to, alkylating agents such as alkyl sulfonates and triazenes. Mechloretamine, cyclophosphamide (Cytoxan®), melphalan (L- Sarcolicin, carmustine (BCNU), lomustine (CCNU), semustine (Me Chill-CCNU), streptozocin, chlorozotocin, uracil mustard, chlorme Chin, ifosfamide, chlorambucil, pipobromane, triethylenemelamine, trie Examples include lelenthiophosphoramine, busulfan, dacarbazine, and temozolomide. .

[0226] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminate. This includes, but is not limited to, enzyme inhibitors such as cytarabine (CYTOSAR-U) and cytosine. Arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-th Oguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propagyl-5,8-dideazafolate (PDD F, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucobol Examples include fludarabine phosphate, pentostatin, and gemcitabine.

[0227] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics) (including, but not limited to, enzymes, lymphokines, and epipodophyllotoxins), Ara- C, Paclitaxel (Taxol(registered trademark)), Docetaxel (Taxotere(registered trademark)) Registered trademarks)), deoxycoformycin, mitomycin-C, L-asparaginase, A Zathioprine; Brekinal; Alkaloids, e.g., vincristine, vinblastine , vinorelbine, vindesine, etc.; podophyllotoxins, e.g., etoposide, teniposide Antibiotics such as anthracyclines, daunorubicin hydrochloride (daunorubicin) (Rubidomycin, Serubidin), Idarubicin, Doxorubicin, Epirubicin, and Morpholino derivatives, etc.; phenoxyzomides cyclopeptides, e.g., dactynomycete n; basic glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., Plicamycin (mitramycin); anthracendione, e.g., mitoxantrone; Azilinopyrroindelegone, e.g., mitomycin; macrocyclic immunosuppressants, e.g., Contains cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin, etc. It can be done.

[0228] Other antiproliferative cytotoxic agents include navelben, CPT-11, anastrazole, and letra. Zol, capecitabine, reloxafine, cyclophosphamide, ifosamide, and doro It is xafin.

[0229] Microtubule-inhibiting agents with antiproliferative activity are also suitable for use, but are not limited to, aloko Ruchtine (NSC 406042), Halichondrin B (NSC 609395), Col Chicin (NSC 757), colchicine derivatives (e.g., NSC 33410), dorus Tachin 10 (NSC 376128), Meitansin (NSC 153858), Rizoki Syn (NSC 332598), Paclitaxel (Taxol®), Taxo l(registered trademark) derivatives, docetaxel (Taxotere(registered trademark)), thiocolchic acid (NSC 361792), tritylcisterine, vinblastine sulfate, vincris Ethin sulfate, natural and synthetic epothyron (etopyrone A, epothyron B, discoderm) This includes, but is not limited to, lidocaine, estramustine, and nocodazole.

[0230] Suitable hormone regulators and steroids (including synthetic analogs) for use include Adrenoco Luticosteroids, such as prednisone and dexamethasone, estrogen and pre Gestin, for example, hydroxyprogesterone caproate, medroxyprogesterone acetate Adrenal gland, megestrol acetate, estradiol, clomiphene, tamoxifen, etc. Cortical inhibitors, for example, aminoglutethimide, 17α-ethinylestradiol, dieth Rustilbestrol, testosterone, fluoxymesterone, dromos propionate Tanolone, Testactone, Methylprednisolone, Methyl-testone, Prednisolone Lon, triamcinolone, chlorotrianicene, hydroxyprogesterone, aminoglucan Tethymide, Estramustine, Medroxyprogesterone acetate, Leuprolide, Flutamide This includes do (drogenil), toremifene (Fairston), and Zoladex, but These are not the only ones. Estrogen stimulates proliferation and differentiation, and therefore estrogen Compounds that bind to receptors are used to block this activity. (e.g., corticosteroids) This can inhibit T cell proliferation.

[0231] Other chemotherapeutic agents include metal complexes, such as cisplatin (cis-DDP) and carbop. Latin, etc.; urea, e.g., hydroxyurea; and hydrazine, e.g., N-methyl hydrazine. Drazin; epidophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxa This includes throne, leucovorin, tegafur, etc. Other antiproliferative agents of interest include immunosuppressants. Epidemic control agents, for example, mycophenolic acid, thalidomide, desoxysperguarine, azath Polin, Leflunomide, Mizoribine, Azaspiran (SKF105685); Ires sa (registered trademark) (ZD1839, 4-(3-chloro-4-fluorophenylamino)- This includes 7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline, etc. .

[0232] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. It contains "paclitaxel" (e.g., docetaxel, TAXOL(trademark), TA). XOTERE (trademark) (docetaxel formulation), 10-desacetyl derivatives of paclitaxel Similar compounds and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl compound of paclitaxel. (This should be understood to include analogs, formulations, and derivatives such as analogs.) This refers to the art known to those skilled in the art (WO94 / 07882, WO94 / 07881, WO94 / 0 7880, WO94 / 07876, WO93 / 23555, WO93 / 10076, USA Patent No. 5,294,637, No. 5,283,253, No. 5,279,949, Same No. 5,274,137, No. 5,202,448, No. 5,200,534, Same No. (See Sections 5,229,529 and EP590,267) It is possible, or for example, Sigma Chemical Co., St. Louis, Mo.(T7402 from Taxus brevifolia, or Taxus yan It can be obtained from various commercial sources, including T-1912 from nanensis.

[0233] Paclitaxel is available in a variety of forms, including the commonly available chemical forms of paclitaxel, as well as other similar forms. Similar compounds and derivatives (e.g., Taxotere® docetaxel mentioned above) and paclita Paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextrose) Proteins such as , paclitaxel-xylose, or Abraxane®. It should be understood that this refers to conjugated paclitaxel.

[0234] The term "taxane" encompasses a wide range of derivatives, including both hydrophilic and hydrophobic derivatives. This also includes derivatives of taxanes. International Patent Application No. WO99 / 18113 concerns derivatives of taxanes. Galactose and mannose derivatives described in [the same document], and the P [the same document] described in [the same document] WO99 / 14209. Perazino and other derivatives, Nos. WO99 / 09021, Nos. WO98 / 22451, and derivatives of taxanes described in U.S. Patent No. 5,869,680, WO98 / 28288 The 6-thio derivative described in [reference], the sulfenamide described in U.S. Patent No. 5,821,263 This includes derivatives, as well as taxol derivatives as described in U.S. Patent No. 5,415,869. , but not limited to these. Paclitaxel prodrugs are also included, WO98 / 589 27, WO98 / 13059, and the items described in U.S. Patent No. 5,824,701 This includes, but is not limited to, these items.

[0235] Useful immunotherapies include anti-PD-1 / PD-L1 immunotherapy and / or treatment methods. Immunotherapy drugs that can be targeted include, for example, CTLA-4, LAG-3, and TIM-3. This includes other immunotherapies that target markers such as anti-PD-1 / PD-L1. Immunotherapy, for example, involves administering an effective dose of one or more anti-PD-1 / PD-L1 antagonists to a target. This includes, but is not limited to, administering antagonists, For example, OPDIVO(registered trademark) (nivolumab), KEYTRUDA(registered trademark) (pe Ambrolizumab, Tecentriq (trademark) (atezolizumab), durvalumab MEDI4736), avelumab (MSB0010718C), BMS-936559 ( MDX-1105), CA-170, BMS-202, BMS-8, BMS-37, BM Examples include, but are not limited to, S-242. These antibodies are provided herein. It may be administered as combination therapy with the provided anti-ABCG2 antibody.

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

[0237] LAG-3 is currently undergoing clinical trials to treat cancer. Anti-LAG-3 immunotherapy includes: Activates T effector cells (transmits LAG-3 inhibitory signals to pre-activated LAG-3+ cells) (By downregulation) induced (i.e., antigen-specific) Treg inhibitory activity This includes methods that use an antagonist LAG-3 antibody that can both inhibit and suppress the antagonist. LAG-3 antagonist antibodies include relatrimab (BMS-986016, Brist). Developed by ol-Myers Squibb, IMP701 (Immutep Developed by TESARO, Inc., TSR-033 (anti-LAG-3mAb, TESARO, Inc.) This includes (developed by...).

[0238] Immunotherapy also includes, for example, adoptive cell therapy (ACT) and chimeric antigen receptor (CAR) therapy. This includes T-cell-based immunotherapies such as T-cell therapy. For example, the target is the cancer. A population of CAR T cells that have been engineered to target antigens expressed in this way may be administered. T-cell-based therapies have been used in some cases to obtain blood samples or tumor biopsies from subjects. Obtaining cell samples and ex vivo culturing immune cells from the samples, and the genetics of the cultured immune cells. This may involve culturing regardless of whether or not there is sub-modification. For example, immune cells may be the target. These were obtained, cultured ex vivo, and modified with CARs specific to the antigen expressed by the cancer. This can produce a population of CAR T cells. Then, the CAR T cells are reintroduced into the target. This can then target cancer. T cell-based immunotherapy depends on the specific cancer being treated. For example, by targeting various antigens, various cell types can be collected / cultured. It can be composed in various ways, such as by [method]. In addition, T cell-based immunotherapy is, for example, For example, by intravenous injection or locally, for instance, by injection (e.g., intraperitoneal injection, pleural injection) It can be administered systemically by methods such as direct injection (e.g., via tetrathrombus infusion).

[0239] In some cases, the treatment methods described herein apply to multidrug-resistant tracheostomies. This includes, but is not limited to, porters, such as multidrug resistance transporters other than ABCG2. This may include administering one or more inhibitors, which are not specified. Useful inhibitors include, for example, tyrosine kinase inhibitors, natural products, microRNAs, and This includes small molecule inhibitors. Inhibitors of multidrug-resistant transporters include ABC transporters. It contains a protein inhibitor.

[0240] Individuals suitable for treatment using the method disclosed herein include individuals with cancer, and individuals diagnosed with cancer. Individuals that have been sterilized, individuals being treated for cancer with chemotherapy, radiation therapy, antibody therapy, surgery, etc. , to cure cancer (for example, with one or more of the following: chemotherapy, radiation therapy, antibody therapy, surgery, etc.) Individuals who have been treated but are not responding to treatment, cancer (e.g., chemotherapy, radiation therapy) They have been treated with one or more of the following: therapies, antibody therapy, surgery, etc., but have not responded to the initial treatment. However, this includes individuals whose cancer subsequently relapsed, i.e., those whose cancer returned.

[0241] The methods disclosed herein are applicable, for example, to primary cancer, secondary cancer, recurrent cancer, recurrent cancer, and refractory cancer. It can be used to target and treat various cancers, including sexually transmitted cancers. For example, several In that case, the method of this disclosure was used as the initial treatment for the primary cancer identified in the subject. It can be used in some cases. In some cases, the method of this disclosure may be used in a manner other than primary (for example, secondary or As a treatment (from then on), for example, in patients with cancer that is refractory to prior treatment, In subjects with cancer that has grown regrowth after prior treatment, mixed response to prior treatment (e.g., , positive response to at least one tumor in the subject and at least one in the subject In subjects with a negative or neutral response to the second tumor, etc., it is used in such cases. It is possible.

[0242] In some cases, the method disclosed herein is used for drug-resistant cancers such as multidrug-resistant cancers. It can be used to treat the target. Multidrug resistance (MDR) is a condition in which many cancers are resistant to chemotherapy drugs. This mechanism develops resistance to the drug, resulting in minimal cell death and the expansion of drug-resistant tumors. MDR cancer may be accompanied by one or more resistance mechanisms, such as an increase in efflux pumps. This can lead to reduced expression, decreased drug absorption, inhibition of cell death or apoptosis, and regulation of drug metabolism. This includes, but is not limited to, the methods described herein that, in some cases, prevent MDR. It can be stopped, reversed, or avoided.

[0243] In some cases, the method of this disclosure is used to treat cancers that are resistant to the first drug. The target is subjected to an effective amount of the target described herein when combined with a second drug different from the first drug. This may include treatment with antimicrobial antibodies. For example, in some cases, the target cancer It may be resistant to the first chemotherapy agent, and the target is a second chemotherapy agent different from the first. By administering an effective amount of the target antibody described herein in combination with the chemotherapeutic agent... It can be treated. Various combinations of first and second chemotherapy agents can be used for treatment, for example. It can be used depending on the type of insect, the potential for developing resistance, etc.

[0244] Many cancers are known to develop drug resistance. For this reason and other reasons... Therefore, the method disclosed herein may be approved for use in various cancers, for example, acute lymphoblastic cancer. Leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (examples) For example, Kaposi's sarcoma, lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratogenicity / Rhabdoid tumor, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, bone cancer (for example, Yui (e.g., sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brainstem glioma, brain tumors Examples include gravidoloma, central nervous system embryonic tumor, central nervous system germ cell tumor, craniopharyngioma, epithelioma, etc. , breast cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), bronchial tumors, Burk's Trimphoma, carcinoid tumors (e.g., pediatric, gastrointestinal, etc.), unknown primary tumors, heart ) Tumors, central nervous system (e.g., atypical teratogenic tumors / rhabdoid tumors, germ tumors, germ cell tumors) (Sores, lymphomas, etc.), cervical cancer, childhood cancer, spinal cord tumor, chronic lymphocytic leukemia (CLL), Chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharynx Tumors, cutaneous T-cell lymphoma, ductal tumors (e.g., bile ducts, extrahepatic tumors), non-invasive ductal sclerosing sclerosing (DCIS) , germ tumor, endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, head Extralatinous germ cell tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, eye cancers (e.g., intraocular melanoma, reticular cholangiocarcinoma) (e.g., spleen blastoma), fibrous histiocytoma of the bone (e.g., malignant osteosarcoma), gallbladder cancer, stomach ( gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational trophoblasts Sexually transmitted diseases, gliomas, hair cell leukemia, head and neck cancer, organ cancer, hepatocellular carcinoma (liver cancer), histiocytes Diseases (e.g., Langerhans cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Islet cell tumors (e.g., pancreatic neuroendocrine tumors), Kaposi's sarcoma, kidney cancer (e.g., renal cell tumors) Wilms' tumor, pediatric renal tumors (including nephrocytosis), Langerhans cell histiocytosis, laryngeal cancer , leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia) (CLL, chronic myeloid leukemia (CML), hair cell carcinoma, etc.), lip and oral cancer, liver cancer (primary) (Primary), non-invasive lobular carcinoma (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), Pancreases (e.g., AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary) (Central nervous system (CNS), etc.), macroglobulinemia (e.g., Waldenström's disease) ), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelial cancer Tumor, metastatic cervical squamous cell carcinoma of unknown primary origin, median ductal carcinoma with nut gene, oral cancer, Multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplastic syndrome , myelodysplastic / myeloproliferative neoplasms, myeloid leukemia (e.g., chronic (CML)), myeloid Leukemia (e.g., acute leukemia (AML)), myeloproliferative neoplasms (e.g., chronic tumors), nose Cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cavity Cancer, oral cancer (e.g., lip cancer), oropharyngeal cancer, osteosarcoma, and malignant fibrous histiocytoma of bone. Ovarian cancer (e.g., epithelial, germline, low-grade tumors), pancreatic cancer, pancreatic neuroendocrine cancer Secretory tumors (pancreatic islet cell tumors), papillary tumors, paranasal sinus tumors, paranasal sinus and nasal cavity cancers, parathyroid gland Penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuroblastoma, primary central nervous system (C) NS) Lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and urethral migratory cells Cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, tumors (e.g., Ewing's, Kaposi's, osteosarcoma, Rhabdomyosarcoma, soft tissue, uterus, etc.), Sézary syndrome, skin cancer (e.g., childhood, melanoma, Merkel cell carcinoma, non-melanoma, etc., small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma Hmm, flat neck cancer (for example, primary cancer of unknown origin, metastatic cancer, etc.), stomach (stomach) (Gastric) cancer, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureteral and renal pelvis cancer, urethral cancer, uterine cancer ( For example, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström's hypertension This includes, but is not limited to, cytoglobulinemia and Wilms' tumor.

[0245] The treatment methods described herein, in some cases, have previously been one or more conventional treatments It may be performed on subjects receiving the treatment. For example, in the case of oncology, the method described herein may be In some cases, for example, conventional chemotherapy, conventional radiotherapy, conventional immunotherapy This may follow conventional cancer therapies, including but not limited to, procedures and surgery. In a few cases, the methods described herein apply to subjects who have not responded to conventional therapies, Alternatively, it may be used in cases of refractory disease. In some cases, the method described herein may be used. This may be used when the subject is responding to conventional therapy.

[0246] In some cases, the method disclosed herein is used to identify the minimal residual disease remaining after precancerous treatment. MRD (Medical Reaction Disease) can be used to target, treat, or eliminate the subject. Targeting, treating, and / or eliminating MRD depends on whether the MRD is refractory to prior treatment. Nevertheless, this method may be used to pursue the objectives. In some cases, the method of this disclosure may be used to pursue the objectives. Using this method, MRD can be treated with other methods besides prior treatment or treatment using the multispecific antibodies described herein. After a determination that MRD is refractory to one or more available treatment options, the patient is considered eligible for MRD. They can be targeted, treated, and / or eliminated.

[0247] In some cases, this method can be used preventively for surveillance purposes. For example, the subject who needs it may not have a detectable disease, but for example, drug resistance If you are at risk of developing recurrent cancer, including sexually transmitted cancer, use the single or multiple treatments described herein. Treatment involving one or more specific antibodies may be administered. In some cases, prevention The approach is to identify subjects that are predicted to be drug-resistant or that will become drug-resistant. It may be used when there is a particularly high risk of developing a primary cancer that is expected to occur. In this case, the preventive approach is that the subject has been previously treated for cancer and has not experienced a recurrence. Alternatively, it may be used when there is a risk of developing drug resistance.

[0248] In some cases, the method disclosed herein relates to the expression of one or more markers or therapeutic targets. This may involve analyzing cancer. For example, in some cases, the method is to target By analyzing cancer samples, cancers exceeding a predetermined threshold in ABCG2, and cancers exceeding a predetermined threshold... Erucan-associated antigen or MDR-1, or ABCG2 and TAA exceeding a predetermined threshold This may involve determining whether both, or both ABCG2 and MDR1, are expressed.

[0249] In some cases, whether a subject is treated with the antibody of this disclosure is determined by ABCG2 expression assessment. The outcome may depend on cancer-related antigen expression, or both. For example, in some cases, When cancer expresses ABCG2 above a predetermined threshold, the target is the anti-ABCG2 antibody of this disclosure. Alternatively, the cancer may be treated with the multispecific antibodies of this disclosure, where the cancer expresses ABCG2 below a predetermined threshold. In such cases, the subject may be treated with the multispecific antibody of this disclosure without being treated with the anti-ABCG2 antibody. ru.

[0250] Any convenient assay can analyze ABCG2 and / or cancer-related antigen levels. It can be used in, for example, flow cytometry, nucleic acid-based assays (e.g., amplification, This includes, but is not limited to, sequencing, cell cytometry, immunohistochemistry, etc. Any suitable biological sample may be used, for example, a cancer biopsy sample, but this Not limited to these, but useful for evaluating the expression of one or more markers and / or targets. A fixed threshold can be determined by any convenient and appropriate method, and the measured expression level and This includes comparison with a corresponding control. For example, in some cases, the sample was assayed. Useful predetermined thresholds for ABCG2 and / or cancer-related antigen levels are used in healthy / normal ranges. This can correspond to the levels of ABCG2 and / or cancer-associated antigens measured in reference cells such as cells. .

[0251] Method of production As summarized above, the methods of this disclosure also enable the production and / or production of antibodies described herein. This includes methods for identification. The target antibody can be identified by any known method, e.g., for protein synthesis. It can be produced by conventional synthesis methods, recombinant DNA methods, etc.

[0252] If the target antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. It can be synthesized using [a specific method]. When polypeptides are chemically synthesized, the synthesis is carried out in the liquid phase. Alternatively, the process may proceed via a solid phase. The C-terminal amino acid of the sequence is bound to an insoluble support, and then... Solid-phase polypeptide synthesis (SPPS), in which the remaining amino acids are sequentially added to the sequence, is used to target antibodies. This is an example of a suitable method for the chemical synthesis of the body. Various forms of SPPS such as Fmoc and Boc. However, it can be used to synthesize the target antibody.

[0253] Standard recombinant methods can be used to produce the target antibody. For example, optional recombinant methods Nucleic acids encoding the light chain and heavy chain variable regions, which are linked to the constant region, are inserted into the expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segment encoding the immunoglobulin chain is an immunoglobulin polypeptide. In an expression vector that ensures the expression of [the gene], it is manipulably linked to a control sequence. The column contains the promoter (e.g., a naturally occurring or heterologous promoter), the signal sequence, and the This includes, but is not limited to, enhancer elements and transcription termination sequences. It also includes expression regulatory sequences. This involves transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). It can be a eukaryotic promoter system in a vector that can be used. When the vector is incorporated into a suitable host, the host exhibits high levels of expression of the nucleotide sequence. Furthermore, it is maintained under conditions suitable for antibody collection and purification.

[0254] Due to the degeneracy of the genetic code, various nucleic acid sequences code each immunoglobulin amino acid sequence. It can be done. The desired nucleic acid sequence can be synthesized by de novo solid-phase DNA synthesis, or by the desired port. Polymerase chain reaction (PCR) mutations of variants prepared faster than inucleotides It can be generated by induction. Oligonucleotide-mediated mutagenesis is a target polypeptide mutagenesis. This is an example of a preferred method for preparing substitution, deletion, and insertion variants of ptyide DNA. See Adelman et al., DNA 2:183 (1983). Simply put, the target polypeptide DNA is an oligonucleotide that encodes the desired mutation. DNA is modified by hybridizing it with a single-stranded DNA template. After the procedure, DNA polymerase is used to incorporate the oligonucleotide primers. Synthesize the entire second complementary strand of the template and encode the selected modifications within the target polypeptide DNA. To make someone do it.

[0255] Suitable expression vectors are typically expressed as episomes or as an indispensable expression of host chromosomal DNA. As a missing portion, it can be replicated within the host organism. Generally, an expression vector is used to express the desired DNA. Selective markers (e.g., Ampici) that enable the detection of these cells transformed with sequences. Phosphorus resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neo Contains mycin resistance.

[0256] Escherichia coli cross-links the polynucleotide encoding the target antibody. Examples of prokaryotic host cells that can be used for training. Other suitable for use Its microbial hosts include rod-shaped bacteria such as Bacillus subtilis, and Salmonella. This includes Serratia and other Enterobacteriaceae such as various Pseudomonas species.

[0257] Other microorganisms, such as yeast, are also useful for expression. (e.g., Saccharomyces, Sc) Erubiae and Pichia are examples of suitable yeast host cells, and if desired, A suitable vector having a current control sequence (e.g., promoter), replication origin, termination sequence, etc. It is accompanied by 3-phosphoglycerate kinase and other glycolytic enzymes. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. It includes. Inducible yeast promoters include, in particular, alcohol dehydrogenase, i Promotion of socytochrome C, as well as enzymes involved in the utilization of maltose and galactose. It includes tar.

[0258] In addition to microorganisms, mammalian cells (for example, mammalian cells grown in in vitro cell culture) Using the present invention (for example, polynucleotides encoding immunoglobulins or fragments thereof), It is also possible to express and produce polypeptides of Otido. Winnacker, Fro m Genes to Clones, VCH Publishers, NY,N. See Y. (1987). Suitable mammalian host cells include CHO cell lines, and various others. Cos cell lines, HeLa cells, HEK cells, myeloma cell lines, and transformed B cells or ibridomas are one example. Expression vectors for these cells contain expression regulatory sequences, for example. The replication origin, promoter, and enhancer (Queen et al., Immuno nol.Rev.89:49(1986)), as well as ribosome binding sites, RNA splatter Necessary processing such as chair sites, polyadenylation sites, and transcription terminator sequences. It may contain an information site. Examples of suitable expression regulatory sequences include immunoglobulin genes, SV 40. Originating from adenovirus, bovine papillomavirus, cytomegalovirus, etc. He is a promoter. Co et al., J.Immunol. 148:1149(1 See 992).

[0259] Once synthesized (either chemically or recombinantly), the entire antibody, its dimer, and individual light antibodies are produced. Chains and heavy chains, or other forms of the target antibody (e.g., scFv), are standard in the art. It can be purified by following a standard procedure, which involves ammonium sulfate precipitation and affinity Column, column chromatography, high-performance liquid chromatography (HPLC) purification, This includes electrophoresis (generally, Scopes, Protein Purification). (See Springer-Verlag, NY, (1982)) The target antibody is substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least approximately 90% to 95% pure, or 98% to 99% or more pure It may contain, for example, contaminants such as cell debris and macromolecules other than the target antibody. stomach.

[0260] In some embodiments, a method for generating multispecific antibodies in this disclosure involves using candidate antibodies This may include producing and screening for activity. By using a series of steps, both ABCG2 and cancer-associated antigens or MDR1 can be expressed. It is possible to generate multispecific antibodies that specifically bind to the cells. The steps of such a method include: Each has an ABCG2 binding domain and a cancer-related antigen binding domain / MDR1 binding domain. To produce multispecific antibodies or multiple antibodies that contain or are expected to contain First test cells expressing ABCG2 and cancer-related antigens or MDR-1 are multispecific. Contact with an antibody or multiple antibodies, and ABCG2 or cancer-associated antigen / MDR1 The second cell expressing either of these is brought into contact with a multispecific antibody or multiple antibodies, and the first The binding of multispecific antibodies to the first cell was compared to the binding of multispecific antibodies to the second cell. The binding specificity ratio is determined, and when the ratio exceeds a predetermined threshold, ABCG2 and As a multispecific antibody that is specific to cells expressing both the related antigen / MDR1, This may include identifying one or more of several antibodies, and for such comparative binding. If a threshold is used, the threshold may vary and may be in a range of 1.5:1 or greater, for example, 2 :1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1 This includes, but is not limited to, ratios such as 50:1 and 100:1.

[0261] Various cells can be used in this manner, including, for example, the cells described herein. , but not limited to these. In some cases, cells expressing only ABCG2 and Antibodies can bind to both cells that express only cancer-related antigens and cells that express only cancer-related antigens. For example, In a few cases, this method, in relation to the above steps, involves the second cell being ABCG2 It expresses but does not express cancer-related antigens, but the method expresses cancer-related antigens, ABCG Procedure 2 may further include contacting a third cell that does not express the antibody with a multispecific antibody.

[0262] In some cases, such methods may use one or more controls, for example, control cells. This includes, but is not limited to, control reagents, etc. Useful control cells include one or more This includes individuals with known expression or lack of known expression of related genes or proteins. Useful control reagents include, for example, control antibodies such as monospecific antibodies against known targets. This may, but is not limited to, the following cases may occur. For example, in some cases, this disclosure may apply to the following: The method involves treating the first cell, the second cell, and / or the third cell with a monospecific anti-ABCG2 antibody. and further comprising contacting with a control antibody selected from monospecific anti-cancer-related antigen antibodies. To obtain. Depending on the specific method used, various other or additional counters may be used as needed. It can be used in this way.

[0263] kit Aspects of this disclosure also include kits. These kits may include, for example, the antibodies described herein, multiplexed. Any combination of specific antibodies, reagents, compositions, formulations, cells, nucleic acids, expression vectors, etc. The kit may include the target antibody, the nucleic acid encoding it, or cells containing the target nucleic acid. It can include one or more of these. The kit can be configured for various purposes, for example , treatment kits (for example, kits containing anti-ABCG2 antibodies or multispecific antibodies, and for example, A kit for producing antibodies (which may include one or more additional activators such as chemotherapeutic agents), This includes kits for screening antibodies, etc.

[0264] The optional components of the kit may change, for example, buffers, protease inhibitors, etc. May include. If the kit contains the target nucleic acid, the nucleic acid may also contain restriction sites, multiple clonins. It may have areas for coating, primer, etc. The various components of the kit are in separate containers. It may be possible, or certain compatible components may be pre-assembled into a single container as desired. It is possible.

[0265] In addition to the above components, the kit includes the components of the kit for carrying out the method. Instructions for using it may be included. Instructions for implementing the target method are generally The information is then recorded on a suitable recording medium. For example, the instruction manual is recorded on a base material such as paper or plastic. It can be printed. Therefore, the instructions may be included as a package insert in the kit or its components. The labeling of the container of the component may be present (i.e., packaging or sub-packaging) (Associated with G). In other embodiments, the instructions are in a suitable computer-readable memory. Media, for example, compact disc read-only memory (CD-ROM), digital bar As electronic storage data files present on satellite discs (DVDs), diskettes, etc. It exists. Furthermore, in another embodiment, although the actual instructions are not included in the kit, the remote From the source, for example, means are provided to obtain the instruction manual via the internet. This embodiment includes an example of a web address where the instructions can be viewed and / or where the instructions are down. This kit includes a web address that can be loaded. As with the instruction manual, to obtain the instruction manual... This method is recorded on a suitable substrate.

[0266] Exemplary, non-limiting aspects of this disclosure The aspects of this subject matter described above, including embodiments, may be described individually or in one or more other aspects or embodiments. It may be beneficial in combination with the application method. Without limiting the above explanation, certain non- A limited aspect is provided below. As will become apparent to those skilled in the art upon reading this disclosure, each individual Each of the numbered aspects is used, or each preceding or succeeding aspect is numbered individually. This can be combined with any of the embodiments to which it is attached. This can be combined with all such combinations of embodiments. It is intended to provide support in combination, as explicitly provided below. Not limited to combinations of the above. Those skilled in the art will be able to proceed without departing from the spirit or scope of the present invention. It will be clear that various changes and modifications can be made.

[0267] The following embodiments are provided as examples, not as limitations. [Examples]

[0268] The following examples provide a complete disclosure and description of methods for preparing and using the present invention to those skilled in the art. It is presented for the purpose of limiting the scope of what the inventors consider to be those inventions. The following experiments are not intended to represent all or only the experiments that were conducted. It is not possible. There is no effort to ensure accuracy regarding the numbers used (e.g., quantity, temperature, etc.). However, a certain degree of experimental error and deviation should be taken into consideration. Unless otherwise specified... The ratio is the ratio by weight, the molecular weight is the weight-average molecular weight, and the temperature is in degrees Celsius. The pressure is atmospheric pressure or approximately atmospheric pressure.

[0269] Common methods in molecular biochemistry and cell biochemistry include Molecular Cloni ng: A Laboratory Manual,3rd Ed.(Sambrook et al., HaRBor Laboratory Press 2001), Sh ort Protocols in Molecular Biology,4th E d.(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 & Loe wy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: L aboratory Procedures in Biotechnology(Do yle & Griffiths,John Wiley & Sons 1998) These disclosures are recognized in any standard textbook and are incorporated herein by reference. Reagents and cloning vectors for methods referred to or related to this disclosure. The cells and kits are manufactured by BioRad, Agilent Technologies, and T hermo Fisher Scientific, Sigma-Aldrich, Ne w England Biolabs(NEB), Takara Bio USA,In c. Commercial vendors such as Addgene, Inc., America n Repositories such as Type Culture Collection (ATCC) It is available.

[0270] Example 1: Generation of antibodies that specifically bind to cells expressing ABCG2 Materials and methods antibody generation Wild-type (WT) human ABCG2 and mutant ABCG2 were used for immunization. We generate various mutants of ABCG2 that constrain the pump in either a closed three-dimensional structure or a closed three-dimensional structure. To generate ABCG2 mutants constrained in an open three-dimensional structure, human and Macac The mutation in *A fascicularis ABCG2* is E221Q. Mutations in human ABCG2 to generate ABCG2 mutants that are constrained by three-dimensional structure are (i) K86M, S87A, (ii) K86M, S87A, Q126A, and / or ( iii) K86M, S87A, Q126A, and R246E. Stable cells expressing 3T3-ABCG2 and C6-ABCG2 are used to convert 3T3 and C6 cells into pM cells. Transfection with ono-hygro-ABCG2 plasmid, followed by 0.2m The product was prepared by selecting either g / ml or 0.5 mg / ml hygromycin. AGCG2 Tan Virus-like particles that present proteins have already been reported (Popova et al. al.2008, J Virol 82(3):1389-1398), AGCG2 genetics Co-administration of 293T cells using plasmids carrying the child and retroviral Gag protein. It was generated by transfection.

[0271] Female mice or rats were subjected to various prime-boosting strategies to ABCG2 (wild type) Human or cynomolgus monkey DNA and / or ABCG2 (wild type or mutant) In some cases, the cells were immunized with expressing cells or virus-like particles for 8-12 weeks. And, using one or more antigens (one or more of WT or variant ABCG2) for immunization. This was used to increase the diversity of the antibodies produced and enhance the immune response. Spleen and lymph node cells from another source were fused with SP2 / 0 myeloma cells (hybridoma). (Technology). The presence of anti-ABCG2 antibodies in the hybridoma supernatant was determined by flow cytometry. Screening was performed on the cells, followed by cell killing (chemosensitization) assays and efflux inhibition assays. Functional activity was screened. CDRs from selected mouse IgG were used. As described below, H via transfection using a standard protocol Mammalian IgG1 skeleton for full-length IgG1 antibody expression and production in EK293 host cells It was cloned into an expression vector.

[0272] Expression vector For the generation of antibody expression vectors, a general one optimized for expression in mammalian cell lines Human IgG1 constant heavy chain or human Ig, each pre-inserted into the recipient expression vector. The heavy and light chain DNA sequences can be configured to inframe one of the G1 kappa constant light chains. The variant region was subcloned. The expressed gene was then fully cloned for high-level gene expression. pCI-neovir using human cytomegalovirus (CMV) rapid early promoter It was cloned into a milk expression vector (Promega). Two antibody chains were used in two different ways. It was cloned into a vector.

[0273] The N-terminal signal sequences from the mouse IgG heavy chain and kappa light chain were extracted, respectively, from the heavy chain and light chain. It was used for secretory expression. The signal peptide is cleaved during expression and retains its intact N-terminus. The end was left intact. In the Fab construct, the C-terminus of the CH1 IgG1 constant region was removed for purification. It was combined with the 6×His tag.

[0274] mAb production Expi293 cells (A14527, Ther) growing when suspended with mammalian expression vectors. (moFisher) polymer-based co-transfection of cells, manufacturer's recommendation The antibody construct was expressed using the method described.

[0275] Approximately 6 days after transfection, cells were collected by centrifugation. In detail, Add 1 ug of total coding DNA per 1 ml of culture to be infected with Opti-MEM (registered Dilute in culture medium (Life Technologies) and Expi in the same culture medium. Enter the fectamine reagent (Life Technologies) for 20 minutes. The mixture was then incubated. Next, the mixture was incubated in Expi293® expression medium (Life Expi293® cells growing in suspension (Technologies) The antibody was added at a concentration of 2.5 million cells / ml in air containing 8% CO2 at 37°C. After 6 days, the antibody composition was... The culture medium containing the building blocks was collected by centrifugation.

[0276] Purification of mAbs To purify antibody forms containing human and mouse Fc, use 10 μg per 1 ml of supernatant. MabSelect (trademark) SuRe (trademark) (GE Healthcare) The collected culture medium was added and stirred overnight at 4°C. The next day, the vacuum manifold unit (P All Life Sciences (USA) uses a 24-well filter plate. Protein A resin was applied in a tank. The resin was washed with PBS, and the antibody was treated with 50 mM phosphate pH. The solution was eluted with solution 3 and neutralized with 10×PBS pH 13.

[0277] Analytical testing of mAbs (GXII reduction and non-reduction) The purity and monomer content of the final protein preparation are measured by Protein Express. Use the LabChip Kit (Perkin-Elmer) according to the manufacturer's instructions. Using Caliper's LabChip GXII, high-throughput analysis is performed to determine the results. The chip was determined. The chip was coated with a polymer solution containing 0.2% SDS and a fluorescent dye. It was automatically primed in place. The destaining channel was primed with SDS and a polymer free of dye. - Filled with solution. To put it simply, proteins in reduced and unreduced states are small bodies Mix the sample (2-5 μL) with or without DDT with the caliper sample buffer. The mixture was prepared by combining the samples. The samples were denatured at 75°C for 5 minutes and centrifuged at 2000g for 3 minutes. The cells were separated and then electrophoresed. The electrophoretic graph was generated using LabChip GXII Touch software. Generated by Perkin Elmer.

[0278] Analytical testing (HPLC) for mAbs High-throughput analysis of the purity and monomer content of the final protein preparation using HPLC. This was determined by size exclusion chromatography (SEC), which uses Infinity 1. 260 Agilent HPLC system, Advancebio SEC 300 A 4.6×300mm, 2.7um(p / n PL1580-5301)(Agile The experiment was conducted using PBS and 400 mM sodium chloride (NT Technologies). Using a mobile phase with a pH of 7.4, injection was performed under uniform concentration elution conditions, and absorption was performed at 280 nm. Detection was performed using luminosity. Quantification is based on the relative area of ​​the detected peaks.

[0279] The target antibody is substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least approximately 90% to 95% pure, or 98% to 99% or more pure It may contain, for example, contaminants such as cell debris and macromolecules other than the target antibody. stomach.

[0280] Transient transfection of 293T cells Optimized PEIPro® transfection protocol (Polyplu Using s), 293T cells were treated with pLenti-C-Myc-DDK-P2A-Puro Transient transfection with human P-glycoprotein-tagged ORF clones in plasmids The DNA and JetPEI® were diluted in culture medium, and then approximately 10 Mix gently for 1 minute. This mixing leads to the formation of the transfection complex, The complex was added directly to the cell culture. Efflux blockade was detected using the multidrug resistance direct dye efflux assay (Mu ltidrug Resistance Direct Dye Efflux Ass Measurements were taken using ay)(Chemicon) according to the manufacturer's protocol.

[0281] Discharge blocking experiment procedure 293T_ABCG2_OX cells were washed several times and placed in phenol red-free DMEM. With a cell density of 2 × 10⁶ cells per 1 ml, 96 wells are available as 50 μl portions / well. The cells were seeded on a plate. The cells were then treated with 50 μl of antibody or the small molecule inhibitor ABCG2-Fumito Remolzin C (FTC) or its tetracyclic analog KO143 is mixed at a final concentration of 1 μM. The cells were then combined. Next, the cells were incubated at 37°C for 1 hour in the presence of 2.5 μM mitoxantrone. The cells were then baited. Next, they were washed twice and finally resuspended in 200 ml of PBS. Toxanthrone fluorescence was measured using excitation at 635 nM and emission at 647 nM. The protocol used to measure outgoing interruptions was Szabo E, et al., PLoS One.2 018 Jan 17;13(1):e0190629, Deeken JF,et a l.,Mol Pharmacol.2009 Nov;76(5):946-56) It is accepted.

[0282] Measurement of monoclonal antibody titer binding to KPG2 The binding titer of recombinant antibody and KPG2 transfectant was measured at approximately 666 nM. The procedure was performed by serial dilution. Antibodies diluted with flow cytometry buffer were then measured on ice. The cells were incubated with the buffer for 30 minutes. They were washed twice with flow cytometry buffer. Next, the bound antibody was diluted 1:200 with flow cytometry buffer and then sampled on ice with cells. Goat anti-human IgG (Jac) fragments labeled with PE and incubated for 20 minutes. Detected by kson ImmunoResearch. Flow cytometry After washing twice with a buffer solution, fluorescence was measured using an Attune NxT flow cytometer. Analyze the data with GraphPad Prism 8.0 software to determine EC50. did.

[0283] Bispecific antibodies were characterized by binding titer measurement and chemotoxicity assays.

[0284] Cell binding assay. Antibody binding to cells was evaluated by flow cytometry. Human Alternatively, 293T cells stably transfected to express cynomolgus monkey ABCG2. Cells (293T_ABCG2_OX) are processed in flow cytometry buffer (PBS + 2% FBS) Wash once with (0.02% sodium azide) and transfer 2× cells to flow cytometry buffer. Resuspend at 10^6 cells / mL and transfer 0.1 mL / mL to a 96-well microtiter plate. Dispensed in a bottle. Should the recombinant antibody be added to the cells at 5 ug / mL to confirm initial binding? The cells were then sequentially diluted with flow cytometry buffer starting from 100 ug / mL. After incubation for 30 minutes, the cells were washed twice with flow cytometry buffer. The combined antibody is PE-labeled F(ab')2 fragment goat anti-human IgG (Jackson Immuno). Detected by noResearch and measured with an Attune NxT flow cytometer. The evaluation was performed. EC50 is calculated as the antibody concentration that yields half of the maximum response.

[0285] Cytotoxic assay. The effect of antibodies against topotecan cytotoxicity is evaluated using ABCG2 expression. 293T cells that have been stably transfected to this state are 293T_ABCG2_ Evaluation was performed using OX cells. Cells were placed in 0.05 mL of assay medium (DMEM + 10% FBS). Cells were seeded in white, flat-bottomed, 96-well tissue culture plates, with 5000 cells per well. Potecan is a test antibody or control antibody at 100 ug / mL (2 × final concentration), or 20 Assay containing uM (2 × final concentration) of the low molecular weight ABCG2 inhibitor fumitremorzin C The culture medium was prepared at a 2× final assay concentration by serial dilution starting from 200 μM. Equivolute ( Add 0.05 mL of topotecan / antibody mixture to a 96-well plate 293T_ABC The cells were added to G2_OX cells. The plate was then incubated in 5% CO2 at 37°C. After 72-96 hours, the plate was equilibrated to room temperature, and Promega(registered trademark) Ce The llTiter-Glo® bioluminescent cell viability assay uses the manufacturer's recommended protocol. Cell viability was evaluated using the method according to the guidelines. Luminescence was measured using a Molecular Device. s(registered trademark) FlexStation(registered trademark) 3 multimode microplate refill Measurements were taken using a meter and analyzed using GraphPad Prism 8.0 software. The half-inhibitory concentration (IC50) is the concentration at which the response (cell proliferation) decreases by 50% (e.g., topotecan or This is the concentration of other chemotherapeutic cytotoxic agents.

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

[0287] method: Cell culture: HT1376 cells were cultured in 10% FBS, 1% penicillin, and 1% streptococcus. The cells were maintained in RPMI medium supplemented with mycin at 37°C and 5% CO2. The cell lines used were It was confirmed to be genuine and negative for mycoplasma.

[0288] Inoculation - 2 × 10 cells diluted with PBS:Matrigel (1:1) 6 The individual was anesthetized by 50 animals. Female SCID-Biege mice aged 5-6 weeks were subjected to sterile conditions using a 27G insulin syringe. It was administered as a subcutaneous injection.

[0289] result Figure 1A shows HEK293 naive cells overexpressing human ABCG2 ("hG2"). HEK293 cells and HEK2 cells overexpressing cynomolgus monkey ABCG2 ("cG2") FACS analysis of the binding of anti-ABCG2 antibodies, G2.65 and G.302, to 93 cells is shown. vinegar.

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

[0291] The anti-ABCG2 antibody G2.302 overexpresses human ABCG2 ("hG2"). The 293 cells and the 293 cells that overexpress cynomolgus monkey ABCG2 ("cG2") were linked. It binds, but not significantly to 293 naive cells.

[0292] Figure 1B shows human A cells expressing the anti-human ABCG2 antibody 5D3 in 3T3 and C6 cell lines. 3T3 cells stably transfected with BCG2 and cABCG2 are used to induce The expressed cynomolgus monkey ABCG2 (cG2) and hABCG2 can be stably transferred. This indicates that it binds to human ABCG2(hG2) expressed by controlled C6 cells. .

[0293] Figures 2A-2B. Binding of anti-ABCG2 antibody to 293 cells overexpressing human ABCG2. Compared to the anti-ABCG2 antibody 5D3, the listed anti-ABCG2 antibodies are effective against ABCG2. It has low affinity. Compared to 5D3, it has lower affinity for ABCG2. BCG2 antibodies (i) are used in cancer cells due to the higher expression levels of G2 in non-cancer cells. Compared to other molecules, it has the potential to be conjugated by cytotoxic molecules that preferentially bind to cancer cells. To produce an antibody that has and / or (ii) A targeted by a bispecific antibody It preferentially binds to cells expressing both BCG2 and the second antigen, while expressing only G2. It is more suitable for creating bispecific antibody molecules that bind significantly less to the target cells. obtain.

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

[0295] Figure 2D shows the binding of the anti-G2 antibody to 293T_ABCG2_OX cells. The dissociation constant is shown. These antibodies are selected from those listed in Tables 2 and 3. That is the case.

[0296] Figure 2E shows the effects of various anti-ABCG2 mAbs on efflux blocking in 293T.G2 cells. The results (EC50 shift) are shown (each of the K D (Listed along with their values).

[0297] Table 3 lists the following characteristics of the anti-ABCG2 antibody: measured by FACS, 293T cells that have been stably transfected to express ABCG2 (293T Binding to _ABCG2_OX), binding affinity to 293T_ABCG2_OX cells ( Binding = "+", Non-significant binding = "-"), Binding with cynomolgus monkey ABCG2 (Binding = " + (significant binding = -), topotecan excretion by 293T_ABCG2_OX cells. Efflux blockade (efflux blockade = "+", non-significant efflux blockade activity = "-"), 293T_ABCG 2. Efficacy of listed antibodies against topothecan-induced cell killing in OX cells (killing) (Injury = "+", non-significant killing = "-"). [Table 4]

[0298] G2.640 and G2.643, anti-G2 antibodies produced in rats, are used in the 293T cell line. It did not show significant binding to human or cynomolgus monkey G2 overexpressed. G2.64 Antibody 0 has the following sequence: JPEG2026053316000042.jpg22165 JPEG2026053316000043.jpg22164 Underlined indicates CD-R.

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

[0300] Figure 2F shows the topotheca cells when 293T_ABCG2_OX cells are exposed to anti-G2 antibody. This shows the measurement of the IC50 of cytotoxicity.

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

[0302] Materials and methods Cell line and cell viability experiments HEK293T, MCF-7, N6ADR, and SKNF7 cell lines expressing PgP Obtained from the American Type Culture Collection. All Cell lines and strains derived therefrom are tested with up to 10% fetal bovine serum (Sigma), non-essential RPMI1640 or DMEM supplemented with amino acids and 2 mmol / L L-glutamine. In a humidified incubator, the temperature was maintained at 37°C and 5% CO2 (unless otherwise indicated). (Unless otherwise). The cells were used in the same condition as supplied, or with excess (Ox)PgP. PgP expression is either modified to express PgP or expressed via lentiviral-mediated short hairpin RNA. He was knocked down (KD), or essentially as reported (Cong, L .et al.(2013)Science 339,819-823)CRISPR / Functional genes were knocked out (KO) using Cas-mediated knockout technology and supplied. For IC50 determination of vincristine and paclitaxel, cells were placed in standard growth medium. Sowed the seeds and let them adhere overnight. Paclitaxel, vincristine, or topotecan (Sigm a) was added in a dilution series, and any modifier was added in the range of 1 to 500 μM / L. Survival rates were measured using the Celltiter-Glo bioluminescent cell viability assay (Promega). The drug was used and measured 72 hours later. The concentration of the drug that caused a 50% inhibition of cell viability (IC5) was measured. 0) is multiparameter curve analysis (GraphPad Prism software Gr Calculated from aphPad Software, Inc., and determined after a minimum of two iterations. In most of the experiments conducted, cell survival was observed in response to drug and / or modifier treatment. Cell lines that did not show a 50% decrease in rate were considered to have not reached IC50 by definition. For paclitaxel or the drug / modulator combination under investigation, >1000 nmol / It is listed as having IC50 of L.

[0303] We also created recombinant cell lines that stably express the described monoclonal antibodies (mAbs). .

[0304] Cell culture technology and antibody production Standard cell culture techniques are described in Current Protocols in Cell B. iology(2000), Bonifacino, J.S., Dasso, M., Ha rford, J.B., Lippincott-Schwartz, J. and Yam. Listed in ada, KM (eds.), John Wiley & Sons, Inc. It is used in such a way.

[0305] 293 and CHO cells were subjected to transient nasal injection of mAb, Fab'2, Fab, and bispecific mAb. Used for sex production. Expi293 cells (A14527, ThermoFishe Using polymer-based co-transfection of r), different antibody constructs are expressed. The cells were grown in suspension with a mammalian expression vector, according to the manufacturer's recommendations. Set.

[0306] In the preparation of bispecific antibody molecules, the corresponding expression vector is set to 1:1:4 (heavy chain KK:heavy chain). Cells were transfected using the DD (light chain) ratio. In standard antibody expression, the ratio is 1:2. The ratio (heavy chain:light chain) was used.

[0307] Six days after transfection, cells were collected by centrifugation. In detail, Add 1 μg of total coding DNA per 1 ml of culture to be septic, Opti-MEM (registered Dilute in (Trademark) Culture Medium (Life Technologies) and Expif in the same medium. Ink with ectamine reagent (Life Technologies) for 20 minutes The mixture was then incubated in Expi293® expression medium (Life Expi293® cells growing in suspension (Technologies) The antibody construct was added at a concentration of 2.5 million cells / ml in air with 8% CO2 at 37°C. After 6 days, the antibody construct was... The culture medium containing [the substance] was collected by centrifugation.

[0308] Reagent cells used to test binding, efflux blockade, and cell sensitivity to chemotherapeutic agents. KK Human embryonic kidney (HEK) cell line HEK 293FT (Life Technologi es) 10% fetal bovine serum (HyClone), 2 mM GlutaMAX (Lif e Technologies), 100 U / mL penicillin, and 100 g / mL stoichiometric In Dulbecco's Modified Eagle Medium (DMEM) supplemented with leptomycin, 5% CO2 in The temperature was maintained at 37°C using incubation.

[0309] Optimized PEIPro® transfection protocol (Polyplu Using s), 293T cells were treated with pLenti-C-Myc-DDK-P2A-Puro Transient transfection with human P-glycoprotein-tagged ORF clones in plasmids After diluting the DNA and JetPEI® in the culture medium, for about 10 minutes... They were gently mixed. This mixing led to the formation of a transfection complex, The drug was directly added to the cell culture. Efflux blockade was detected using a multidrug-resistant direct dye efflux assay (Chemical Measurements were taken using (on) according to the manufacturer's protocol.

[0310] Sources of target sequences, antibody sequences, and specific anti-target antibody sequences P-glycoprotein (PgP), also known as multidrug resistance protein 1 (MDR1), pL (Genes ABCB1)(NM) in enti-C-Myc-DDK-P2A-Puro _000927) Tagged ORF clones, and ABCG2 in pCMV6-XL5 The ABCG2(NM_004827) human-tagged ORF clone was obtained from Origene. Anti-CD47 antibody (CC2C6, Seiffert M, et al. (1999) Blo od 94:3633) is from Biolegend, an anti-ABCB1 JSB-1 (MAB 4120) is from Millipore, and anti-human ABCG2 antibody (clone 5D3) is from R& Obtained from D Systems. MDR1 is also referred to as KPB1 in this specification. ABC G2 is also referred to as KPG2 in this specification.

[0311] Generation of stable ABCB1 overexpression (Ox) cell lines To characterize both binding and in vitro efficacy, ABCB1 is stably overexpressed. We developed a cell line that was used in the American Type Culture Collection. Adherent 293T naive cells obtained from ion(ATCC) were used. Commercially available ABCB Flow cytometry using one antibody (Biolegend, clone 4E3.16) When characterized by this, this cell line intrinsically emits ABCB1 at low to moderate levels on its cell surface. This is demonstrated. Using the Polyplus PEIpro reagent, 293T naive cells were AB Transfected with CB1. Three days after transfection, hygromycin B solution Cells were selectively placed using a solution (Millipore Sigma). Continuous high Fourteen days after selection of Glomycin B, 293T cells were evaluated for ABCB1 cell surface expression. This ensures that untransfected cells do not proliferate in subsequent cultures. Therefore, bulk ABCB1-positive 293T cells using fluorescence-activated cell sorting (FACS) Sorting was performed using FACSAria I (BD Biosciences). Selected 293T ABCB1 overexpressing cells were grown, and then ABCB1 overexpressing cells were further processed. I double-checked it.

[0312] Generation of stable ABCG2 overexpressing (Ox) cell lines To characterize both binding and in vitro efficacy, ABCG2 is stably overexpressed. We developed a cell line that was used in the American Type Culture Collection. Using adherent 293T, 3T3, and C6 naive cells obtained from ion(ATCC) The flow chart used was based on a commercially available ABCG2 antibody (R&D System, clone 5D3). When characterized by cytometry, this cell line intrinsically contains ABCG2 on its cell surface. It is expressed at low to moderate levels. 293T naive cells were prepared using lipofectamine 3000 reagent. ABCG2 is transfected, while 3T3 and C6 are neon electroporation Transfected using a method. Three days after transfection, hygromycetes Cells were selected using solution B (Millipore Sigma). Fourteen days after hygromycin B selection, the transfected cells were placed on the surface of ABCG2 cells. Expression was evaluated. Stable ABCG2 cells were treated with FACSAriaI (BD Bio). Using (sciences), single selection and propagation are performed, followed by re-confirmation of ABCG2 overexpression. I acknowledged it.

[0313] Generation of a stable ABCB1 KD 293T cell line To characterize both binding and in vitro efficacy, stable ABCB1 expression was observed. We developed a cell line containing the quaddown gene. R8.74 helper plasmid, VSVG envelope GE Dharmaco contains a pe plasmid and shRNA for ABCB1. By transfection using the GIPZ lentiviral vector, Lentiviruses were generated in 293T naive cells. Then, the collected lentiviruses were used. Adhering 293T naive cells were transduced using this method. Three days after transduction, 293T transducing The introduced cells were subjected to flow cytometry (Biolegend, clone 4E3.16) We evaluated ABCB1 cell surface expression. Compared to naive 293T cells, transduced 293T cells express ABCB1. It did not contain GFP. In addition, the GIPZ lentiviral vector contains GFP. All transduced 293T cells were GFP+, and as expression decreased, the transduction process was completed. It was shown to be successful. The absence of ABCB1 expression was observed in subsequent passages by flow cytometry. Therefore, it was reconfirmed.

[0314] Generation of KO cell lines To construct the ABCG2 gene knockout HEK293 cell line, HEK293 cells The chief cells were first treated with DMEM (Dalbe) supplemented with 10% (v / v) FBS and glutamine. Gibco Modified Eagle Medium, Gibco, Grand Island, NY, USA The cells were cultured via adherent culture. The cells were cultured at 37°C using 5% CO2 at saturated humidity. .

[0315] gRNA design can be done using CRISPR / Cas9, CRISPR / Cpf1, or TALE. To select target sites for N-directed mutagenesis, use the online CHOPCHOP tool. This was done using a boutol. (Kornel Labun et al., (2016)) Nucleic Acids Research, and Tessa G. Montag ue et at.,(2014)Nucleic Acids Res.42:W40 See 1-W407). All designed gRNAs were chemically synthesized (The rmoFisher).

[0316] Use CRISPRMax reagent (ThermoFisher) according to the manufacturer's protocol. Transfection of 293T cells using lipid-based transfection was performed. The procedure was performed. To put it simply, the adherent cells were well-treated one day before transfection. Per cell: 0.2 × 10 5 Individual seeds were sown in 96-well plates. Transfection On this day, GeneArt Platinum Cas9 protein, gRNA, and tran The infection reagent solution was added to the cells. 72 hours after transfection, the limit was reached. Following single cell selection by dilution, cell culture was continued for two weeks in a 96-well plate format. Next, the collected clones were subcultured into 24-well plates, and Guide-it k it(Takara) was tested by genotyping using the manufacturer's protocol. We conducted an experiment. We PCR-amplified the genomic region surrounding the CRISPR target site for each gene. Furthermore, gene editing can alter a single allele in a clone that has been isolated on its own. Determine which of the two alleles (bialleles) gave rise to the indel. The expression of the target protein on clones with mutations in both alleles was performed using FACS. Tested by [method / method].

[0317] Constructing the sequence of the tested molecule (human Fc, mouse Fv, or human Fc or humanized Fv) Expression vectors: Optimized for expression in mammalian cell lines for the generation of antibody expression vectors. Each of the commonly used recipient expression vectors contains pre-inserted human IgG1 stationary weights. Either the heavy chain or the human IgG1 kappa constant light chain, and the heavy chain and light chain DNA sequences in frame. The variable region was subcloned. The gene that should be expressed was used for high-level gene expression. Therefore, pCI- The two antibody chains were cloned into a neo mammalian expression vector (Promega). It was cloned into different vectors.

[0318] The N-terminal signal sequences from the mouse IgG heavy chain and kappa light chain were extracted, respectively, from the heavy chain and light chain. It was used for secretory expression. The signal peptide was cleaved during expression, leaving the intact N-terminus intact. In the Fab construct, the C-terminus of the CH1 IgG1 constant region was treated with 6× to facilitate purification. It was merged into the His tag.

[0319] Regarding the generation of bispecific antibody vectors, the bispecific molecule derived from IgG1 has two distinct properties. It specifically binds to a target, namely the pair PgP(ABCB1) and ABCG2. It includes at least two antigen-binding regions that can be used for binding. Each antigen-binding region has a variable region and a fixed region. This is a Fab fragment consisting of a heavy chain and a light chain, including a normal region. It forms pairs to form Fab anti-PgP( A Fab that is acceptable for both aPgP and Fab anti-ABCG2 (aABCG2) A common light chain capable of causing pairing has been identified, and its use avoids mispairing of LCs. This made it possible. A bispecific construct was fabricated based on the electrostatic steering effect. For example, Gunasekeran et al, (2010) Journal of B See Iological Chemistry 285, 19637-19646. (The entire disclosure is incorporated herein by reference.) In short, the target The polypeptide chain or semi-antibody, on the other hand, undergoes two processes through charge pair substitution in the CH3 domain. It is assembled as a hemispecific antibody, meaning that one of the heavy chains consists of K392D and K409D. One compound contained substitution ("DD"), while the other contained E356K and D399K substitution ("KK").

[0320] Both bispecific mAb molecules are derived from the anti-MDR1 antibody 15D3 on human IgG1Fc. Variable heavy chain containing HCDR1-3, containing HCDR1-3 from anti-G2 antibody G2-255 It contained a variable heavy chain and a common light chain kappa sequence derived from the anti-MDR1 antibody MRK16.

[0321] Previously produced monoclonal antibody 15D3 against PgP (e.g., U.S. Patent No. 5 See Patent Nos. 959,084, the disclosures thereof are incorporated herein by reference in their entirety. (and MRK16 (Iwahashi et al., Cancer Research) (rch 53, 1993, its disclosure is incorporated herein by reference in its entirety) Noclonal antibodies are used as recombinant genetically modified antibodies in human IgG1 / kappa expression vectors. —I cloned it.

[0322] Variable heavy and light chain fragments from mouse hybridoma sequences are available, and leader units The columns and steady-state regions were cloned into the same background.

[0323] The anti-ABCG2 monoclonal antibody sequences are disclosed in Table 2, and include the antibody variable heavy chain and light chain. The strand fragments have the same background in the leader sequence and constant region in two separate vectors. I cloned it into the lands.

[0324] result Detection of specific binding of ABCB1 and ABCG2 The binding specificity of mAbs, Fabs, and bispecific IgG1 is excessively high for human ABCB1 targets. 293T naive cells that express ABCG2, 293T naive cells that overexpress human ABCG2 It was tested using FACS. Briefly, different cell lines were tested with varying amounts of m Ab or bispecific mAb, or human IgG1 isotype control antibody on ice The cells were incubated for 1 hour. The cells were then basted three times in FACS buffer (PBS containing 0.5% BSA). Washed. Alexa647-labeled goat anti-human antibody was added as a secondary antibody, and the sample was heated on ice. The sample was incubated for another hour. The sample was washed and then processed using BD FACS Canto (BD B The analysis was performed using iosciences.

[0325] The binding specificity of mAb, Fab, and bispecific IgG1 was determined for the 293T cell line and human AB. 293T naive cells overexpressing the CB1 target, and 293T naive cells overexpressing human ABCG2. 93T cells were used for testing by FACS. Briefly, different cell lines were tested. Various amounts of mAb or bispecific mAb, or human IgG1 isotype control antibody and Both were incubated on ice for 1 hour. Cells were then incubated in FACS buffer (containing 0.5% BSA). Washed three times with BS. Alexa647-labeled goat anti-human antibody was added as a secondary antibody. The sample was incubated on ice for an additional hour. The sample was washed and then subjected to BD FACS Cant. Analysis was performed using o (BD Biosciences).

[0326] In this embodiment, one arm is connected to the discharge pump MDR1 / PgP, and the other arm is We will demonstrate the construction of a bispecific heterobivalent antibody molecule that binds to the efflux pump ABCG2. When both targets are present on the cell surface simultaneously, bispecific antibodies exhibit relatively high affinity / a It binds to cells via viditi. Compared to MDR1 / PgP or ABCG2, If either is absent or substantially reduced, the binding of bispecific antibodies is significantly reduced. It decreases or becomes undetectable.

[0327] Bispecific antibodies bind to transporter proteins (efflux pump PgP) and... It includes one arm that tagonizes, making cells more sensitive to chemotherapeutic agents. At times, the other arm is connected to another discharge pump ABCG2.

[0328] Example 3: Bispecific antibody that binds to ABCG2 and EGFR This example shows antibodies that can bind to the extracellular domains of ABCG2 and EGFR. To demonstrate the development of this method, EP blockade resensitizes and kills cells that are resistant to chemotherapy agents. To bring about.

[0329] The materials and methods are the same as those described in Example 2. Optimized PEIPro Using the (Trademark) Transfection Protocol (Polyplus), 293T fine Cells were tagged with ABCG2 (NM_004827) human tags in the pCMV6-XL5 plasmid. Transient transfection was performed using an RF clone. Regarding the generation of a bispecific antibody vector. The IgG1-derived bispecific antibody molecule targets two different targets: ABCG2 and EGFR. It includes at least two antigen-binding moieties that can specifically bind to the antigen. These are Fab fragments consisting of heavy chains and light chains, each containing a variable region and a constant region. Therefore, it is acceptable as both Fab anti-EGFR and Fab anti-ABCG2 (aABCG2). A common light chain capable of facilitating bonding was identified.

[0330] Figure 5 shows that both ABCG2 and EGFR are expressed in A549 cells. S analysis. Cells conjugated with PE anti-human ABCG2 antibody, clone 5D3, and PE Staining was performed with conjugated anti-human EGFR antibody, clone AY13.

[0331] The variable heavy and light chain fragments of the anti-ABCG2 KNJY-G2-65 antibody are two distinct vectors. In the test, the leader sequence and the constant region were cloned into the same background.

[0332] The sequence and corresponding structure of the anti-EGFR antibody 6B3S (cetuximab) are shown in the following document: ps: / / www.ncbi.nlm.nih.gov / Structure / pdb / 6B3S(Molecular Basis for Necitumumab Inh ibition of EGFR Variants Associated with Acquired Cetuximab Resistance,Bagchi A, Haidar JN, Eastman SW, Vieth M, Topper M, Ia colina MD, Walker JM, Forest A, Shen Y, Novo siadly RD,Ferguson KM,Mol.Cancer Ther.(2 Available on page 521-531 of 018)17, the array is reproduced here for reference. It will be done. The variable heavy chain sequence of the 6B3S antibody is as follows: The variable light chain sequence of the JPEG2026053316000045.jpg211656B3S antibody is as follows: JPEG2026053316000046.jpg17165F .

[0333] result Detection of EGFR and ABCG2 specific binding The binding specificity of EGFR-targeting mAbs, Fabs, and bispecific IgG1 is determined by rh Using EGFR-his tagged protein (Sino Biological) Tested by ISA. In short, a microtiter plate was subjected to PBS. Coated with 50 μl of purified human HGFR-his protein at 2 μg / ml, Next, blocking was performed with 0.4% BSA in 100 μl of PBS. Dilutions of different antibody forms were also performed. The solution was added to each well with a 1 / 3 serial dilution and incubated at room temperature for 1 hour. FR antibody 6B3S was used as the positive control, and human IgG1 was used as the isotype control. Next, the plate was washed three times with PBS / Tween, and then HRP conjugate was applied. The saturated donkey was incubated with a secondary reagent specific to the human constant region for 1 hour at room temperature. After purification, the plate was developed with HRP substrate. The reaction was stopped with 2M H2SO4, and OD was... The measurement was taken at 520 nM.

[0334] The binding specificity of mAb, Fab, and bispecific IgG1 is demonstrated by overexpressing human ABCG2. 293T naive cells, as well as naive A cells that spontaneously express EGFR and ABCG2. 549 was used and tested by FACS. Briefly, different cell lines were tested. Various amounts of mAb or bispecific mAb, or human IgG1 isotype control antibody and The cells were incubated on ice for 1 hour. Washed three times with S). ​​Alexa647-labeled goat anti-human antibody was added as a secondary antibody. The sample was incubated on ice for another hour. The sample was washed and then processed using BD FACS Canto. Analysis was performed using (BD Biosciences).

[0335] Figure 6 shows the results for two different bispecific mAbs that bind to EGFR and ABCG2. Combined data is provided. aEGFR indicates 6B3S, an anti-EGFR antibody. Bispecificity Antibody: aEGFR DD HC / G2.173KK HC / G.173 LC is DD Variable heavy chain from anti-EGFR antibody 6B3S fused to human IgG1 having a variable, and KK A variable heavy chain from the anti-ABCG2 antibody G2.173 fused to human IgG1 having a conversion, Two copies of the variable light chain of the anti-ABCG2 antibody G2.173 fused to the kappa sequence, include. Bispecific antibody: aEGFR DD HC / G2.65KK HC / G.65 L C is a variable heavy chain from 6B3S fused to human IgG1 with a DD substitution, and a KK substitution. A variable heavy chain from the anti-ABCG2 antibody G2.65 fused to human IgG1, and a cut It includes two copies of the variable light chain of the anti-ABCG2 antibody G2.65 fused to the P sequence.

[0336] In this embodiment, one arm is connected to the discharge pump ABCG2, and the other arm is connected to the EGF. We demonstrate the construction of a bispecific heterobivalent antibody molecule that binds to the R target. Both targets are cell When present simultaneously on the surface, bispecific antibodies exhibit relatively high affinity / avidence. It binds to cells. As shown in Figure 6, the G2.6.5-based bispecific antibody is shown. It has several advantages in binding studies, including a stronger affinity for EGFR. Stronger binding to A549 cells (by ELISA) and both targets presented. This demonstrates that when the ABCG2 efflux pump is overexpressed, both bispecific antibodies are the same To bind together in a specific way.

[0337] Figure 7 shows human ABCG2 ("hG2") and cynomolgus monkey ABCG2 (cG2). The images show the binding of the anti-ABCG2 monoclonal antibody to the overexpressing 293T cells. As indicated by the dissociation constant (Kd), the antibodies G2.748, G2.757, G2.758, and G2.760 binds to both human and cynomolgus monkey ABCG2 with excellent binding affinity. ru.

[0338] Figure 8 shows the interaction between human or cynomolgus monkey ABCG2-overexpressing 293T cells and anti-ABCG The binding of antibody G2.748 was compared with that of anti-ABCG2 antibody 5D3, and the corresponding dissociation constant was determined. This is shown along with Kd. As the Kd value indicates, G2.748 is human and cynomolgus monkey AB It binds to both CG2 molecules with excellent binding affinity.

[0339] Figures 9-11 show 293T cells overexpressing human and cynomolgus monkey ABCG2, respectively. The binding of various recombinant anti-ABCG2 antibodies (Panel No. 1 to No. 3) to and the corresponding It shows a Kd value. Of particular note is the antibody against both human and cynomolgus monkey ABCG2. This is a strong bond between isomers G2.636, G2.631, and G2.643.

[0340] Figures 12 and 13 show small molecule ABCG2 inhibition following the previously described efflux blockade experimental procedure. The agents Fumitremorzin C (FTC) and Ko143 were used as positive controls for excretion. The results of testing the listed recombinant anti-ABCG2 antibodies regarding their inhibitory activity are shown below. In particular, the anti- Body G2.636, G2.643, G2.333, G2.631, and G2.318 are strong They exhibit strong efflux inhibitory activity, with G2.636 and G2.643 being particularly effective.

[0341] Figure 14 shows 293T cells stably transfected to express ABCG2. Anti-AB cells against topotecan cytotoxicity in 293T_ABCG2_OX cells The effects of CG2 antibodies G2.643, G2.420, and G2.631 were investigated by FTC and 5D3. This is shown using as a positive control. All three antibodies tested showed cytotoxic activity of topotecan. This significantly increased [the value].

[0342] Figure 15 shows the anti-ABCG2 antibodies G2.343, G2.636, and G2.629, and the topo The combination with tetracytes is used in xenotransplantation using topotecan-resistant Panc1 / T300 cells. In a single-dose trial, it was shown to be effective in reducing tumor volume. PANC1(ATC C, CRL-1469) is a pancreatic ductal epithelial carcinoma cell line. Topotectan-resistant Panc1 / T300 cells increase in topotheca levels of 25, 50, 100, 200, and 300 nM. By continuously incubating PANC1 cells in a culture medium containing a certain concentration... The cells were developed. The selected cells showed elevated ABCG2 expression and were different from the parent PANC1 cells. In comparison, it was resistant to topotecan. The arrows indicate the administration schedule.

[0343] Figure 16 shows the combination of anti-ABCG2 antibodies G2.343 and G2.631 with topotecan. However, non-small cell lung cancer (NSCLC) epithelial cancer cell line A549 (ATCC, CCL-18 In xenograft studies using 5), it was shown to be effective in reducing tumor volume. The arrows indicate the administration schedule. All combinations reduce tumor volume, but G2 The .343 / topotecan combination has been shown to be particularly effective.

[0344] Figure 17 shows the non-small cell lung cancer (NSCLC) epithelial cancer cell line A549 (ATCC, CCL). In xenograft studies using -185), camptothecin-11 (CPT) alone or in combination with other methods, 11. Efficacy of anti-ABCG2 antibody G2.333 administered in combination with irinotecan. The arrows indicate the administration schedule. The efficacy of the G2.33 / CPT11 combination is This significantly exceeds the effectiveness of the 5D3 / CPT11 combination in this experiment.

[0345] Figure 18 shows the results of a xenograft test using topotecan-resistant Panc1 / T300 cells. Bispecific anti-ABCG2 antibody G2 was administered alone or in combination with topotecan. The efficacy of 318 / KT3 / G2.318 is shown. The arrows indicate the administration schedule. KT3 = Cetuximab, an anti-EGFR antibody. The bispecific antibody tested was used as a monotherapy and as part of a combination therapy. Both combinations with Potecan significantly reduced tumor volume.

[0346] Figure 19 shows the results using the HT1376 (ATCC, CRL-1472) bladder epithelial carcinoma cell line. In xenograft studies, the bispecificity of administration either alone or in combination with topotecan. This shows the efficacy of the anti-ABCG2 antibody G2.318 / KT9 / G2.318. The arrows indicate the administration schedule. The Joules are shown. KT9 = atezolizumab, an anti-PD-L1 antibody. The bispecificity tested was The combination of antibodies and topotecan has been shown to be effective in reducing tumor volume. It is.

[0347] Figure 20 shows various humanized G2.636 anti-ABCG2 antibodies against human and cynomolgus monkey ABCG2. It exhibits efflux inhibitory activity and binding to G2. Anti-ABCG2 antibody G2.636 is effective in all human populations. The variant retains activity, and the G2.636.hu47 variant exhibits particularly excellent expression. It exhibits activity.

[0348] Figure 21 shows two humanized ABCG2 / CD47 bispecific antibodies (5F9huscFv- Summary of G2.318.hu33 and B6H12huscFv-G2.318.hu33 The structure, and its binding to human and cynomolgus monkey ABCG2, respectively, are described in G2.31. 8. Compared with hu33 and 5D3, both humanized antibodies show excellent binding.

[0349] Figure 22 shows the humanized ABCG2 / HER2 bispecific antibody KT1scFv-G2.318. The general structure of .hu33, and its binding to human ABCG2 and human HER2 respectively. This indicates that KT1=HER2. The bispecific antibody is potent against both ABCG2 and HER2. It shows a bond.

[0350] Figures 23A-23C show the bispecific antibodies G2.318KK KT9DD G2.318. Outline structure, and ABCG2+KT9-(Figure 23A), ABCG2-KT9+(Figure 23B) ), and its binding to ABCG2+KT9+293T cells (Figure 23C) is shown. 9 = Atezolizumab, an anti-PD-L-1 monoclonal antibody. The antibody is ABCG2 and It shows excellent binding to both PD-L1.

[0351] Figures 24A and 24B show the G2.643 antibody and its humanized form -G2.643.hu46. The binding of to human and cynomolgus monkey ABCG2 (Figure 24A), as well as G2.643 and G 2.643. Shows the efflux inhibitory activity of the hu46 antibody (Figure 24B).

[0352] The aforementioned inventions are described in some detail as examples and embodiments for clarity of understanding. However, without departing from the intent or scope of the attached claims, certain modifications and It will be readily apparent to those skilled in the art, in light of the teachings of the present invention, that modifications can be made.

[0353] Therefore, the above merely illustrates the principles of the present invention. Those skilled in the art will find the express provisions of this specification to be useful. Although not described or shown, the principles of the present invention are embodied and included within its spirit and scope. Please understand that various configurations can be devised. Furthermore, all of the listed specifications Examples and conditional language are primarily provided by the inventors to promote the principles and techniques of this invention. This is intended to assist readers in understanding the concepts being discussed, and such specific enumeration is intended to help them understand the concepts being discussed. It should be interpreted that this is not limited to examples and conditions in which the present invention is applicable. Furthermore, the principles of the present invention All descriptions in this specification that enumerate aspects and embodiments, as well as specific examples thereof, are related to their structure. It is intended to encompass both tactile and functional equivalents. In addition, such equivalents are currently Both known equivalents and future-developed equivalents, i.e., the same machine regardless of structure. It is intended to include any developed elements that perform the function. Furthermore, disclosed herein Any such disclosure is not explicitly enumerated in the claims. Nevertheless, it is not intended for public consumption.

[0354] Therefore, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. This is not the case. Rather, the scope and spirit of the present invention are defined by the appended claims. This is manifested in the claims, 35 U.S.C. §112(f) or 35 U.S. .SC§112(6) requires that the exact phrase "of" be placed at the beginning of such limitation in the claims. Only if the phrase "means for" or the exact phrase "steps for" is included, Explicitly defined as being invoked to limit the scope of the claim, and such exact phrasing If the terms are not used to limit the scope of the claims, 35 U.S.C. §112(f ) or 35 U.S.C. § 112(6) shall not be invoked.

Claims

1. Antibody that specifically binds to ATP-binding cassette subfamily G member 2 (ABCG2). The body, and the antibody, with respect to binding to ABCG2, Table 2 lists the heavy chain phases of pairs of antibody variable heavy chain (VH) and variable light chain (VL) regions. Includes complementarity-determining regions 1-3 (HCDR1-3) and light chain CDR1-3 (LCDR1-3) An antibody that competes with another antibody.

2. The claim according to claim 1, comprising HCDR1 to HCDR3 of the VH region of the antibody listed in Table 2 above. antibody.

3. The claim 2 includes LCDR1 to 3 of the VL region of the antibody listed in Table 2 above. antibody.

4. Table 2 lists the heavy chain phases of pairs of antibody variable heavy chain (VH) and variable light chain (VL) regions. The antibody according to claim 1, comprising a complementarity-determining region (HCDR) and a light chain CDR (LCDR).

5. Antibody that specifically binds to ATP-binding cassette subfamily G member 2 (ABCG2). Body molecule, wherein the antibody has a variable heavy chain (VH) region and a variable light chain of the antibodies listed in Table 2. Heavy chain complementarity determination regions 1-3 (HCDR1-3) and / or light chain CD in pairs with the (VL) region. Antibody molecules containing R1-3 (LCDR1-3).

6. The aforementioned antibody is a pair of HCDR1-3 and VL regions of the antibody listed in Table 2. The antibody molecule according to claim 5, comprising LCDR1 to 3.

7. Claim that the antibody comprises HCDR1 to HCDR3 of the VH region of the first antibody listed in Table 2. The antibody described in item 5.

8. Claim 7, wherein the antibody comprises LCDR1 to 3 of the VL region of the second antibody in Table 2. The antibody molecule described.

9. The antibody molecule is the variable light (VL) chain and / or variable weight (VH) chain of the antibody listed in Table 2. The antibody molecule according to claim 5, comprising a chain.

10. When the antibody binds to a cell expressing ABCG2, the efflux by ABCG2 is suppressed. An antibody molecule according to any one of the prior claims that inhibits.

11. The antibody molecule according to any one of the prior claims, wherein the antibody comprises a humanized light chain.

12. The antibody molecule according to any one of the prior claims, wherein the antibody comprises a humanized heavy chain.

13. The antibody is a bispecific antibody, Ig monomer, Fab fragment, F(ab') ２ Fragment, Fd A prior claim selected from the group consisting of fragment, scFv, scAb, dAb, and Fv An antibody molecule as described in any one of the items.

14. Claims 1 to 1, wherein the antibody comprises a VL region and a VH region present in a separate polypeptide. An antibody molecule as described in any one of the 12 items.

15. Claims 1 to 1, wherein the antibody comprises a VL region and a VH region present in a single polypeptide. The antibody molecule described in any one of item 2.

16. ATP-binding cassette subfamily G member 2 (ABCG2) and multidrug resistance proteins A bispecific antibody molecule that binds to 1 (MDR1), wherein the antibody molecule is two identical It includes a variable light (VL) chain, a first variable heavy (VH) chain, and a second VH chain, wherein the VL chain is Each contains an antigen-binding site for MDR1, and the first VH chain is an antigen-binding site for MDR1. The second VH chain includes a primordial binding site, and the second VH chain includes an antigen-binding site for ABCG2, The second VH chain, when paired with one of the light chains, binds to ABCG2, double Specificity antibody molecule.

17. The first VH chain has the amino acid sequence: EVKVVESGGVLVRPGGSLKLSC A. １ TYYP DSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGA GDAWFAYWGQGTLVTVS (Sequence No. 418) (X here) １ is N, Q, or Heavy chain complementarity determining regions 1-3 of the VH chain of an anti-MDR1 antibody (HCDR) having S) 1-3) includes, or The first VH chain is at least 90% relative to the amino acid sequence shown in SEQ ID NO:

418. amino acids having %, at least 95%, at least 99%, or 100% identity A bispecific antibody molecule according to claim 16, comprising a sequence.

18. The first VH chain is the heavy chain complementarity determining region 1-3 of the VH chain from the anti-MDR1 antibody (HC DR1-3) are included, and HCDR1 has the sequence: GFTFSRYTMS (SEQ ID NO: 419) Including HCDR2, sequence: VATISSGGGNTYYPDSVKG (SEQ ID NO: 362) ), VATISSGGGQTYYPDSVKG (SEQ ID NO: 363), or VATISSG Contains GGSTYYPDSVKG (Sequence ID 364), and HCDR3 has sequence: ARYGA A bispecific antibody molecule according to claim 16, comprising GDAWFAY (SEQ ID NO: 365).

19. The second VH chain is the heavy chain phase of the VH chain of an anti-ABCG2 antibody having the sequence shown in Table 2. The description in any one of claims 16 to 18, which includes complementarity determination regions 1 to 3 (HCDR 1 to 3). A bispecific antibody molecule.

20. The second VH chain comprises HCDR1-3 of the VH chain of the anti-ABCG2 antibody, and the HCD R1 contains sequence: NNAMS (SEQ ID NO: 82), and HCDR2 contains sequence: TITG The HCDR3 contains GGSYTYYPDSVKG (Sequence ID 112), and the sequence is PD Contains GNYEGVLAY (SEQ ID NO: 154), or The second VH chain has the following amino acid sequence: EVMLVESGGALVKPGGSLKLSCAASGFTFSNNAMSWVRQ TPETRLEWVATITGGGSYTYYPDSVKGRFTISRDNARNTL YLQMSSLRSEDTATYYCASPDGNYEGVLAYWGQGTLVTVS For A (Sequence No. 13), at least 90%, at least 95%, at least 99%, Or any one of claims 16 to 19, comprising an amino acid sequence having 100% identity The bispecific antibody molecule described in the section.

21. The two identical VL chains mentioned above have the amino acid sequence: DVLMTQTPLSLPVSLGDQA SISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRLEAEDLGVYYCFQGSHF Light chain of the VL chain of the anti-MDR1 antibody containing PRTFGGGTRLEIK (SEQ ID NO: 367) A dual-layer according to any one of claims 16 to 20, including CDR1 to 3 (LCDR1 to 3). Specificity antibody molecule.

22. The two identical VL chains mentioned above contain the light chains CDR1-3 (LCDR1-3) of the anti-MDR1 antibody. Including, (i) The LCDR1 is sequence: RSSQSIVHSTGNTYLE (Sequence ID 368 ) including, (ii) The LCDR2 includes sequence: KVSNRFS (Sequence ID 305), (iii) The LCDR3 contains sequence: QGSHFPRT (sequence number 369), or The VL chain has the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLE WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRLEAEDLGVYYCFQGSHFPRTFGGGGTRLEIK (Sequence ID 36) 7) at least 90%, at least 95%, at least 99%, or 100% A dual according to any one of claims 16 to 21, comprising an amino acid sequence having % identity. Specificity antibody molecule.

23. The two identical VL chains mentioned above have the amino acid sequence: DVLMTQTPVSLSVSLGDQA SUBSTANCE SYSYS ２ LOVE LOVE LOVE LOVE LOVE LOVE RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASH FPRTFGGGTKLEIK (Sequence ID 370) (where X) ２ is N, Q, or S The VL chain light chains CDR1-3 (LCDR1-3) of the anti-MDR1 antibody have ( or The VL chain has the following amino acid sequence: DVLMTQTPVSLSVSLGDQASISCRSSQSIVHSTGX ２ TYL EWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGVYYCFQASHFPRTFGGGTKLEIK (Sequence ID 3) 70) (X here) ２ (is N, Q, or S) at least 90%, less Both contain amino acid sequences that are 95%, at least 99%, or 100% identical. or the bispecific antibody molecule according to any one of claims 16 to 20.

24. The two identical VL chains mentioned above contain the light chains CDR1-3 (LCDR1-3) of the anti-MDR1 antibody. Including, (i) The LCDR1 is arranged as follows: RSSQSIVHSTGX ２ TYLE (Sequence No. 37) 1) including, (ii) The LCDR2 includes sequence: KISNRFS (Sequence ID 372), (iii) The LCDR3 includes sequence: FQASHFPRT (sequence number 373), Here, X ２ is N, Q, or S, the double according to any one of claims 16 to 20 Specificity antibody molecule.

25. ATP-binding cassette subfamily G member 2 (ABCG2) and multidrug resistance proteins A bispecific antibody molecule that binds to 1 (MDR1), wherein the antibody molecule is two identical It includes a variable light (VL) chain, a first variable heavy (VH) chain, and a second VH chain, wherein the VL chain is Each contains an antigen-binding site for ABCG2, and the first VH chain is for MDR1 The second VH chain includes an antigen-binding site, and the second VH chain includes an antigen-binding site for ABCG2. The first VH chain, when paired with one of the light chains, binds to MDR1, double Specificity antibody molecule.

26. The first VH chain has the amino acid sequence: EVKVVESGGVLVRPGGSLKLSC A. １ TYYP DSVKGRFTVSRDNAMSSLYLQMSSLRSEDTALYYCARYGA GDAWFAYWGQGTLVTVS (Sequence No. 418) (X here) １ is N, Q, or Heavy chain complementarity determining regions 1-3 of the VH chain of an anti-MDR1 antibody having S (HCDR1- The bispecific antibody molecule according to claim 25, comprising (3).

27. The first VH chain is the heavy chain complementarity determining region 1-3 of the VH chain from the anti-MDR1 antibody (HC DR1-3) are included, and HCDR1 has the sequence: GFTFSRYTMS (SEQ ID NO: 419) Including HCDR2, sequence: VATISSGGGNTYYPDSVKG (SEQ ID NO: 362) ), VATISSGGGQTYYPDSVKG (SEQ ID NO: 363), or VATISSG Contains GGSTYYPDSVKG (Sequence ID 364), and HCDR3 has sequence: ARYGA A bispecific antibody molecule according to claim 25, comprising GDAWFAY (SEQ ID NO: 365).

28. The second VH chain is the heavy chain phase of the VH chain of an anti-ABCG2 antibody having the sequence shown in Table 2. The description in any one of claims 25 to 27, which includes complementarity determination regions 1 to 3 (HCDR 1 to 3). A bispecific antibody molecule.

29. The two identical VL chains mentioned above are the light chain CDRs of the VL chains of the anti-ABCG2 antibodies listed in Table 2. The dual singularity according to any one of claims 25 to 28, including 1 to 3 (LCDR1 to 3). antibody molecule.

30. Claims that the antibody inhibits the activity of at least one of ABCG2 and MDR1. A bispecific antibody as described in any one of items 16 to 29.

31. ATP-binding cassette subfamily G member 2 (ABCG2) and tumor-associated antigen (TA A) A bispecific antibody molecule that binds to two identical variable light ( It comprises a VL (Very Light) chain, a first variable weight (VH) chain, and a second VH chain, Each of the VL chains contains an antigen-binding site for ABCG2, and the first VH chain is A The second VH chain contains an antigen-binding site for BCG2, and the second VH chain contains an antigen-binding site for TAA. Including the bonding site, when the second VH chain is paired with one of the light chains, TAA or Each of the VL chains contains an antigen-binding site for the TAA, and the first VH chain is A The second VH chain contains an antigen-binding site for BCG2, and the second VH chain contains an antigen-binding site for TAA. Including a bonding site, when the first VH chain is paired with one of the light chains, ABCG A bispecific antibody molecule that binds to part 2.

32. Each of the VL chains contains an antigen-binding site for ABCG2, and the first VH chain is It contains an antigen-binding site for ABCG2, and the second VH chain is the antigen for TAA. Including a binding site, when the second VH chain is paired with one of the light chains, TAA A bispecific antibody molecule according to claim 31, which binds to the bispecific antibody molecule.

33. The first VH chain is the heavy chain complementarity determining region 1-3 of the anti-ABCG2 antibody listed in Table 2. A bispecific antibody molecule according to claim 32, comprising (HCDR1-3).

34. The first VH chain includes heavy chain complementarity determination regions 1 to 3 (HCDR1 to 3), and the HC DR1 contains the sequence: DDYVH, and HCDR2 contains the sequence: RIDPANGNTRY APKFRG (SEQ ID NO: 115) is included, and the HCDR3 has the sequence: PLWVGGFAY (Sequence ID 157) is included, or the first VH chain has the amino acid sequence: QVQLQQSGADLVRPGASVKLSCTASGFNIKDDYVHWVKQ RPEQGLEWIGRIDPANGNTRYAPKFRGKATMTADTSSNTA YLQLSSLTSADTAVYYCSPPLWVGGFAYWGQGTLVTVSS( Sequence ID 16), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQ APGQGLEWIGRIDPANGNTRYAPKFRGRATMTADTSISTA YMELSRLRSDDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS( Sequence ID 17), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQ APGQGLEWIGRIDPAQGNTRYAPKFRGRATMTADTSISTA YMELSRLRSDDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS( Sequence ID 18), or EVQLVQSGAEVKKPGASVKVSCKASGFNIKDDYVHWVRQ APGQGLEWIGRIDPASGNTRYAPKFRGRATMTADTSISTA YMELSRLRSDDDTAVYYCSPPLWVGGFAYWGQGTLVTVSS( For Sequence ID No. 19), at least 90%, at least 95%, or 100% are identical. A bispecific antibody molecule according to claim 32 or 33, comprising an amino acid sequence having properties.

35. The antigen-binding sites of the two VL chains are the light chain CDR1-3 of the antibodies listed in Table 2. A bispecific antibody molecule according to any one of claims 32 to 34, comprising LCDR1 to 3). 。

36. The antigen-binding sites of the two VL chains are in the sequence: RSSQSLVHSDVNTYLH( LCDR1 containing sequence number 270, L containing sequence: KVSNRFS (sequence number 305) CDR2 and LCDR3 containing sequence: SQTTHVPYT (SEQ ID NO: 334) , or the VL chain has the following amino acid sequence: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDVNTYLH WYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRVESEDLGIYFCSQTTHVPYTFGGGTKLEIK (Sequence ID 19) 9), or DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDVNTYLH WYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRVEAEEDVGVYFCSQTTHVPYTFGGGTKLEIK (Sequence ID 20) 0) Having at least 90%, at least 95%, or 100% identity A bispecific antibody molecule according to any one of claims 32 to 35, comprising an amino acid sequence.

37. The first VH chain includes heavy chain complementarity determination regions 1 to 3 (HCDR1 to 3), and the HC DR1 includes sequence: SGYIS (sequence number 84), and HCDR2 includes sequence: WIY AGTGISNFNQKFTG (Sequence ID 114) is included, and the HCDR3 is sequence: G ARKTLDF (SEQ ID NO: 156) is included, or the first VH chain has the amino acid sequence: QGQMHQSGAELVKPGASVKLSCKTSGFTFNSGYISWLKQ KPRQSLEWIAWIYAGTGISNFNQKFTGKAQLTVDTSSSSTA YMQLSSLTSADSAIYFCASGARKTLDFWGQGTSVTVSS For column number 15), at least 90%, at least 95%, or 100% identity A bispecific antibody molecule according to claim 32 or 33, comprising an amino acid sequence having the above.

38. The antigen-binding sites of the two VL chains are the light chain CDR1-3 of the antibodies listed in Table 2. A bispecific antibody molecule according to claim 37, comprising LCDR1-3).

39. The antigen-binding sites of the two VL chains are sequence: KASDQINYWLA (SEQ ID NO: 2) LCDR1 containing 69), LCDR2 containing sequence: GATSLET (sequence number 10), and The sequence includes LCDR3 containing QQYWTTPYT (sequence number 333), or the V The light chain has the following amino acid sequence: DIQMTQSSSYLSVSVGGRVTITCKASDQINYWLAWYQQK PGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSFQ For TEDVATYYCQQYWTTPYTFGGGTKVEIK (Sequence ID 198) amino acid mixture having at least 90%, at least 95%, or 100% identity A bispecific antibody molecule according to claim 37 or 38, comprising a column.

40. The TAA is EGFR, and the second VH chain has the amino acid sequence: QVQLQES GPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGL EWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSV TAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS(Sequence No. 3) Includes heavy chain complementarity determining regions 1-3 (HCDR1-3) of the VH chain of the 6B3S antibody, which contains 88). The bispecific antibody molecule according to any one of claims 32 to 39.

41. The first VH chain containing HCDR1-3 as shown in the table below, and the second VH chain containing HCDR1-3 Claim 32 includes a combination of the VH chain and a common VL chain containing LCDR1 to 3. The listed bispecific antibody molecule. Table 1

42. The antibody comprises a humanized light chain, according to any one of claims 16 to 41, with the bispecificity described above. antibody molecule.

43. The antibody comprises a humanized heavy chain, according to any one of claims 16 to 41, for bispecificity. antibody molecule.

44. Any one of claims 1 to 15 for use in a method for treating cancer in a subject The antibody molecule described above, or the bispecific antibody molecule described in any one of claims 16 to 43. The method includes administering the antibody to the subject, and the antibody molecule or Bispecific antibody molecule.

45. The method involves administering the antibody in combination with at least one additional activator. The at least one additional activator is a chemotherapeutic agent, a multidrug-resistant transporter For use according to claim 44, comprising an inhibitor, immunotherapy agent, or combination thereof The antibody molecule or bispecific antibody molecule.

46. The at least one additional activator is a chemotherapeutic agent, and optionally, the chemotherapeutic agent The drugs used are Taxol, Vinca alkaloids, Anthracyclines, Etoposide, Mitoxa An antibody molecule for use according to claim 45, which is thorone or methotrexate. Bispecific antibody molecule.

47. The target of treatment is a cancer that has been determined to be resistant to treatment with the aforementioned chemotherapy agent. An antibody molecule or bispecific antibody molecule for use according to any one of claims 44 to 46, having sexual antibody molecule.

48. A pharmaceutical composition, The antibody according to any one of the prior claims, A pharmaceutical composition comprising a pharmaceutically acceptable excipient.

49. The pharmaceutical composition according to claim 48, further comprising an additional activator.

50. The pharmaceutical composition according to claim 48, wherein the additional activator is a chemotherapeutic agent.

51. The additional active agent includes an inhibitor of a multidrug-resistant transporter, as described in claim 50. A pharmaceutical composition.

52. The pharmaceutical composition according to claim 50, wherein the additional activator comprises an immunotherapy agent.

53. An antibody molecule according to any one of claims 1 to 15, or any one of claims 16 to 43 One or more nucleic acids containing one or more sequences encoding the bispecific antibody molecules described in the section.

54. One or more recombinant expression vectors comprising one or more nucleic acids as described in claim 53.

55. A host genetically modified with one or more recombinant expression vectors according to claim 54. cell.

56. It contains an ABCG2 binding domain, a transmembrane domain, and an intracellular signaling domain. An immune effector cell containing a chimeric antigen receptor (CAR), wherein the ABCG2 binding The domain is the variable heavy chain (VH) region and the variable light chain (VL) region of the antibodies listed in Table 2. Paired heavy chain complementarity determination regions 1-3 (HCDR1-3) and / or light chain CDR1-3 (LCD Immune effector cells, including R1-R3).

57. A method for assaying the expression of ABCG2 on the cell surface of a cell, wherein the cell is... A method comprising contacting an antibody described in any one of items 1 to 15.

58. The method according to claim 57, wherein the antibody is labeled in a detectable manner.

59. A method for inhibiting the efflux activity of ABCG2 expressed by living cells, wherein the cells A method comprising contacting an antibody according to any one of claims 1 to 15.

60. Claim 58 further comprises contacting the cells with an inhibitor of MDR1-mediated efflux. Method of description.

61. The method according to claim 59 or 60, further comprising contacting the cells with a chemotherapeutic agent. Law.

62. The method according to any one of claims 59 to 61, wherein the cells are cancer cells.

63. The method according to claim 62, wherein the cancer cells are multidrug-resistant cancer cells.

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