Antibody-drug conjugate

Abstract

The present invention relates to an antibody-drug conjugate targeting c-Kit and its uses.

Description

Technology Field

[0001] The present invention relates to an antibody-drug conjugate targeting c-Kit and its uses. Background Technology

[0002] When Stem Cell Factor (SCF) binds to c-Kit (also known as CD117), several signaling pathways are activated, including phosphoinositide 3 kinase, phospholipase C-gamma, Src kinase, Janus kinase-signal transducers, and transcriptional activators (Yasuda A et al., Dig Dis Sci 52, 2292-2300. (2007); Sun J, Pedersen M & Ronnstrand L; J Biol Chem 284, 11039-11047. (2009)).

[0003] Previous studies have reported that the expression of c-Kit with oncogenic mutations is unregulated or upregulated in various cancers, leading to SCF-independent c-Kit activation and cell proliferation. Overexpression of c-Kit is induced by hypoxia as well as the activation of various transcription factors, including AP-2, ETS, SP1, MYB, and MITF. Currently, more than 500 c-Kit mutations have been identified in human tumors (Sanger Institute Catalog of Somatic Mutations in Cancer, https: / / cancer.sanger.ac.uk SCF-independent spontaneous activation c-Kit mutations are detected in approximately 85%, 30%, 25%, 25%, and 90% of gastrointestinal stromal tumors (GIST), acute myeloid leukemia, acral melanoma, testicular carcinoma, and systemic mastocytoma (SM) (Lennartsson J & Ronnstrand L, Physiol Rev 92, 1619-1649. (2012)).

[0004] The efficacy of small molecules such as imatinib for eliminating cancer stem cells harboring constitutively activated mutations in c-Kit is limited in tumor cells harboring mutations in different sites. To overcome c-Kit mutation-induced treatment resistance, various small molecules such as dasatinib, sunitinib, and axitinib have been developed (Abbaspour Babaei M et al., Drug Des Devel Ther 10, 2443-2459. (2016)). However, the rate of grade 3 / 4 adverse events in patients treated with these small molecule therapies is higher than in patients treated with imatinib, which limits their effective application in cancer treatment (Kantarjian HM et al., Blood 119, 1123-1129. (2012)).

[0005] Antibody-drug conjugates (ADCs) enable the delivery of potent cytotoxic agents by utilizing the specificity of monoclonal antibodies against cancer cells. Recently, various ADCs targeting CD30 (brentuximab vedotin), CD22 (inotuzumab ozogamicin), CD79b (polatuzumab vedotin), and HER2 (trastuzumab emtansine and trastuzumab deruxtecan) have been approved by the U.S. Food and Drug Administration (FDA) (Leung D et al., Antibodies (Basel) 9, 2. (2020)). Since these ADCs are antibody-based drugs that bind to the extracellular domain of a target molecule, they have the relative advantage of not having mutations that mainly occur in the intracellular domain and can be applied regardless of various mutations. The problem to be solved

[0006] The object of the present invention is to provide an antibody-drug conjugate of the following chemical formula:

[0007] Ab-(L1) m -(S) n -(L2) p -(D) q

[0008] In the above formula,

[0009] Ab is an anti-c-Kit antibody or its antigen-binding fragment that specifically binds to the epitope of human c-Kit at SEQ ID NOs 9 and 10;

[0010] L1 is a linker connecting the above-mentioned Ab and S or Ab and L2;

[0011] S is a spacer in a form where the polymer is bonded or not bonded;

[0012] L2 is a cleavable linker;

[0013] D is a drug moiety;

[0014] m is an integer from 0 to 8;

[0015] n is an integer from 0 to 8;

[0016] p is an integer from 1 to 8;

[0017] q is an integer from 1 to 8.

[0018] Another objective of the present invention is to provide an antibody-drug conjugate of the following formula:

[0019] Ab-(L) x -(D) y

[0020] In the above formula,

[0021] Ab is an anti-c-Kit antibody or its antigen-binding fragment that specifically binds to the epitope of human c-Kit at SEQ ID NOs 9 and 10;

[0022] L is a linker including a cleavable linker;

[0023] D is a drug moiety;

[0024] x is an integer from 1 to 8;

[0025] y is an integer from 1 to 8.

[0026] Another objective of the present invention is to provide a composition comprising the antibody-drug conjugate.

[0027] Another objective of the present invention is to provide a method for preventing or treating cancer, comprising the step of administering a pharmaceutically effective amount of the composition to a subject in need thereof.

[0028] Another objective of the present invention is to provide the therapeutic use of the above composition.

[0029] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate.

[0030] Another objective of the present invention is to provide a use for the manufacture of the above pharmaceutical composition.

[0031] Another objective of the present invention is to provide a composition for diagnosing cancer comprising the antibody-drug conjugate.

[0032] Another objective of the present invention is to provide a method for providing information for the diagnosis of cancer, comprising the step of treating a sample separated from a subject with the antibody-drug conjugate.

[0033] Another objective of the present invention is to have an antibody or its antigen-binding fragment (Ab) that specifically binds to human c-Kit (L1) m -(S) n -(L2) p -(D) q The present invention provides a method for producing an anti-c-Kit antibody-drug conjugate comprising the step of conjugating:

[0034] In the above formula,

[0035] L1 is a linker connecting the above-mentioned Ab and S or Ab and L2;

[0036] S is a spacer in a form where the polymer is bonded or not bonded;

[0037] L2 is a cleavable linker;

[0038] D is a drug moiety;

[0039] m is an integer from 0 to 8;

[0040] n is an integer from 0 to 8;

[0041] p is an integer from 1 to 8;

[0042] q is an integer from 1 to 8.

[0043] Another objective of the present invention is to provide a method for producing an anti-c-Kit antibody-drug conjugate comprising the step of conjugating a linker (L) and a drug (D) to an antibody or its antigen-binding fragment comprising the heavy chain CDR1 of SEQ ID NO. 1, the heavy chain CDR2 of SEQ ID NO. 2, the heavy chain CDR3 of SEQ ID NO. 3, the light chain CDR1 of SEQ ID NO. 4, the light chain CDR2 of SEQ ID NO. 5, and the light chain CDR3 of SEQ ID NO. 6, wherein L is a linker comprising a cleavable linker and D is a drug moiety.

[0044] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0045] The present invention relates to antibody-drug conjugates and their uses, and demonstrates that cancer can be controlled, treated, remissioned, or reduced, or recurrence suppressed, by administering an antibody-drug conjugate comprising, for example, an antibody that specifically binds to human c-Kit or its antigen-binding fragment.

[0046] To facilitate understanding of the present invention, numerous terms and phrases are defined. Additional definitions are provided throughout the detailed description.

[0047] I. Definition

[0048] In this specification, the term “approximately” is used to mean approximately, roughly, around, or within a range. When the term “approximately” is used with a numerical range, it modifies the range by extending the boundary above and below the presented numerical value. Generally, the term “approximately” can modify the numerical value above and below the mentioned value by a variation of up to 10 percent, for example, above or below (higher or lower).

[0049] Furthermore, "and / or" should be interpreted to mean that each of the two specified features or components is specifically disclosed either together with the other or alone. Accordingly, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A (alone)," and "B (alone)." Likewise, the term "and / or" used in phrases such as "A, B and / or C" is intended to include the modes of A, B and C; A, B or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone), respectively.

[0050] As used herein, the terms “treat,” “treating,” and “treatment” refer to any type of intervention or process performed on a subject, or the administration of an activator to a subject, for the purpose of reversing, improving, alleviating, inhibiting, or delaying the progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical indicators associated with the disease. Treatment may be performed on a subject with the disease or on a subject without the disease (e.g., for prevention).

[0051] As used herein, the term “administration” refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent to a subject using any of the various methods and delivery systems known to those skilled in the art. Different routes of administration for the antibody-drug conjugates disclosed herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the term “parenteral administration” generally refers to a mode of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intra-arterial, intravertebral, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and substernal injections and infusions, as well as in vivo electroporation. Alternatively, the antibody-drug conjugate disclosed herein may be administered via a parenteral route, for example, via a topical, epithelial, or mucosal administration route, for example, via a nasal, oral, vaginal, rectal, sublingual, or topical route. Additionally, administration may be performed, for example, once, multiple times, and / or over one or more extended periods.

[0052] As used herein, the term “therapeutic effective dose” refers to an amount of drug, either alone or in combination with other therapeutic agents, that is effective in “treating” a subject’s disease or disorder, or effective in reducing the risk, potential, likelihood, or occurrence of a disease or disorder (e.g., cancer). “Therapeutic effective dose” includes an amount of drug or therapeutic agent that provides some improvement or benefit to a subject who has or is at risk of having a disease or disorder (e.g., cancer). Accordingly, “therapeutic effective dose” is an amount that reduces the risk, potential, likelihood, or occurrence of a disease or disorder, or provides some alleviation or relief and / or reduces at least one indicator (e.g., cancer) and / or reduces at least one clinical symptom of the disease or disorder.

[0053] As used herein, the term "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells within the body. "Cancer" or "cancer tissue" may include a tumor. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade neighboring tissues, which may also metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, distal tumors may be said to have originated from a "pre-metastatic tumor." For example, a "tumor derived from melanoma" refers to a tumor resulting from metastatic melanoma. Since distal tumors originate from a pre-metastatic tumor, "derived from" may also include the pre-metastatic tumor; for example, a tumor derived from melanoma may include melanoma.

[0054] The phrase “inhibits tumor growth” as used herein includes any measurable reduction of tumor growth, e.g., at least about 10%, at least about 20%, 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%, at least about 99%, or up to 100%.

[0055] The terms "effective dose," "pharmaceutical effective dose," or "effective dosage" are defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. The "therapeutic effective dose" or "therapeutic effective dosage" of a drug or therapeutic agent is the amount of drug that, when used alone or in combination with other therapeutic agents, promotes disease regression demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention of impairment or injury caused by the disease. The therapeutic effective dose or effective dosage of a drug includes the "prophylactic effective dose" or "prophylactic effective dosage," which is the amount of drug that, when administered alone or in combination with other therapeutic agents to subjects at risk of developing the disease or suffering from a recurrence of the disease, inhibits the onset or recurrence of the disease. The efficacy of a therapeutic agent that promotes disease regression or inhibits the onset or recurrence of a disease can be evaluated using various methods known to those skilled in the art, for example, by analyzing the activity of the agent in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or in in vitro analysis.

[0056] For example, an anticancer agent is a drug that promotes cancer regression in a target. In some embodiments, a therapeutically effective dose of this drug promotes cancer regression until the cancer is eliminated. "Promotion of cancer regression" means that administering a pharmaceutically effective dose of the drug, either alone or in combination with an antineoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free periods, prevention of disability or injury caused by the disease, or other improvement in the patient's disease symptoms. In addition, the terms "effective" and "effectiveness" include both pharmacological efficacy and physiological safety in relation to treatment. Pharmacological efficacy refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to toxicity levels or other negative physiological effects (side effects) at the cellular, organ, and / or organism level resulting from the administration of the drug.

[0057] For example, in the case of tumor treatment, the therapeutically effective or effective dose of the drug inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to an untreated subject. In some embodiments, the therapeutically effective or effective dose of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by up to 100%. The ability of the inhibitor to inhibit tumor growth can be evaluated using the assay described below. Alternatively, this property of the composition can be evaluated by testing the ability of the inhibitor to inhibit cell growth, and such inhibition can be measured in vitro by assays known to those skilled in the art. In other embodiments disclosed herein, tumor regression may be observed and may persist for at least about 20 days, at least about 40 days, or at least about 60 days.

[0058] Some aspects of the present invention relate to the diagnosis of cancer in a subject.

[0059] As used herein, the term “diagnosis” refers to a method that may be used to determine or predict whether a patient is suffering from a given disease or condition. A person skilled in the art may make a diagnosis based on one or more diagnostic markers (e.g., expression levels of c-Kit), wherein the presence, absence, amount, or change in amount of the diagnostic marker indicates the presence, severity, or absence of the said condition. In some embodiments, an increase in the expression of c-Kit in biological samples from a subject is an indicative of a tumor. The term “diagnosis” does not mean the ability to determine the presence or absence of a specific disease with 100% accuracy, nor does it mean that a given course or outcome is more likely to occur than not. Instead, a person skilled in the art will understand the term “diagnosis” as meaning an increased probability that a specific disease is present in the subject.

[0060] The term "diagnostic marker" (e.g., c-Kit expression) refers to a substance capable of diagnosing a tumor by isolating it from normal cells, and includes organic biomolecules, e.g., polypeptides, or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which increase or decrease in tumor cells. The diagnostic marker disclosed herein for cancer may be a protein expressed from the gene for c-Kit, which is increased in tumor cells.

[0061] A composition for diagnosing cancer comprises a preparation for measuring the expression level of mRNA of the c-Kit gene or the amount of expressed protein. Such preparation comprises an oligonucleotide having a sequence complementary to c-Kit mRNA, a primer or nucleic acid probe that specifically binds to c-Kit mRNA, and an antibody or its antigen-binding fragment that specifically binds to the c-Kit protein (hereinafter referred to as an anti-c-Kit antibody) or an antibody-drug conjugate in which an anti-c-Kit antibody is linked to a drug via a linker.

[0062] As used herein, the term “object” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0063] The term "c-Kit" belongs to class III of receptor tyrosine kinases (RTKs) and is also known as the receptor of SCF.

[0064] "c-Kit" comprises any variant or isoform of c-Kit naturally expressed by cells. Accordingly, the anti-c-Kit antibody or antibody-drug conjugate disclosed herein may cross-react with different isoforms of the same species (e.g., different isoforms of human c-Kit) or with c-Kit of a non-human species (e.g., mouse c-Kit). Alternatively, the anti-c-Kit antibody may be specific to human c-Kit and may not exhibit cross-reactivity with other species. c-Kit, or any variant and isoform thereof, may be isolated from cells or tissues that naturally express them or may be generated recombinantly. The sequences of domains 1 to 3 of human c-Kit (Q26 to D309), excluding 25 signal peptides, may be SEQ ID NO. 12.

[0065] The term “antibody” is a term of the art and may be used interchangeably herein, and refers to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the term includes the whole antibody and any antigen-binding fragment (i.e., “antigen-binding portion”) or short chains thereof. In one embodiment, “antibody” refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or the antigen-binding portion thereof. In another embodiment, “antibody” refers to a short-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region consists of three domains CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain CL.

[0066] The VH and VL regions can be further subdivided into a supervariable region called the complementation determining region (CDR), which is interspersed with more conserved regions called the framework region (FR). Each VH and VL consists of three CDRs and four FRs, which are arranged from the amino-terminus toward the carboxy-terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the authentic complement system.

[0067] The antibody may have any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any type (e.g., IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subtype (e.g., IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and IgG3 in mice). Immunoglobulins, e.g., IgG1, exist in several allotypes, which differ from each other by up to a few amino acids. The antibody disclosed herein may be derived from any of the commonly known isotypes, types, subtypes, or allotypes. In certain embodiments, the antibody disclosed herein is an IgG1, IgG2, IgG3, or IgG4 subtype or any hybrid thereof. In certain embodiments, the antibody is of a human IgG1 subtype or a human IgG2 or human IgG4 subtype.

[0068] "Antibody" refers, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, total synthetic antibodies; short-chain antibodies; monospecific antibodies; multispecific antibodies (including bispecific antibodies); tetramer antibodies containing two heavy chains and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; antibody light chain-antibody heavy chain pairs; intrabodies; heteroconjugated antibodies; monovalent antibodies; camelized antibodies; affibodies; It comprises a single-domain antibody (sdAb) comprising an anti-idiotype (anti-Id) antibody (e.g., including an anti-anti-Id antibody) and a binding molecule comprising a single monomer variable antibody domain (e.g., a VH domain or a VL domain) capable of sufficiently binding to an antigen (Harmen MM and Haard HJ Appl Microbiol Biotechnol. 77(1): 13-22 (2007)).

[0069] As used herein, the term “antigen-binding portion” of an antibody, i.e., “antigen-binding fragment,” refers to one or more fragments of an antibody having the ability to specifically bind to an antigen (e.g., domain 2 / 3 of the human c-Kit). Such “fragments” are, for example, about 8 to about 1500 amino acid lengths, suitably about 8 to about 745 amino acid lengths, more suitably about 8 to about 300, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acid lengths. It has been found that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments included in the terms “antigen-binding portion” or “antigen-binding fragment” of antibodies, e.g., the anti-c-Kit antibodies disclosed herein, include, but are not limited to: (i) a Fab fragment which is a monovalent fragment composed of VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment which is a divalent fragment comprising two Fab fragments connected by a disulfide bridge at a hinge region; (iii) a Fd fragment composed of VH and CH1 domains; (iv) a Fv fragment composed of VL and VH domains of a single arm of the antibody, and a disulfide-linked Fvs(sdFv); (v) a dAb fragment composed of a VH domain (Ward et al., (1989) Nature 341: 544-546); and (vi) a separated complementarity determining region (CDR) or (vii) a combination of two or more separated CDRs that may be optionally led by a synthetic linker. Additionally, two of the Fv fragment The domains VL and VH are encoded by separate genes, but they can be joined using recombinant methods via synthetic linkers, thereby allowing the VL and VH regions to pair up to form a single protein chain that constitutes a monovalent molecule (referred to as single-chain Fv (scFv)) (e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc.(See Natl. Acad. Sci. USA 85:5879-5883). These short-chain antibodies are also included within the terms “antigen-binding portion” or “antigen-binding fragment” of the antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. The antigen-binding portion may be produced by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulin.

[0070] The terms “variable region” or “variable domain” as used herein are commonly used interchangeably in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which differs significantly in sequence from the antibody and are used because of the binding and specificity of the antibody to a specific antigen. Sequence variability is concentrated in a region called the complementarity determining region (CDR), and a more highly conserved region within the variable domain is called the framework region (FR).

[0071] The CDRs of the light and heavy chains are believed to be primarily responsible for the interaction and specificity of antigens and antibodies. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes a rodent or murine CDR and a human framework region (FR). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes a rodent or murine CDR and a primate (e.g., non-human primate) framework region (FR).

[0072] The term “heavy chain” as used herein, when used in relation to antibodies, may refer to any different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, each of which gives rise to the IgA, IgD, IgE, IgG and IgM types of antibodies, including subtypes of IgG, e.g., IgG1, IgG2, IgG3 and IgG4.

[0073] As used herein, the term “light chain” may refer to any different type, e.g., kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain when used in relation to antibodies. Light chain amino acid sequences are well known in the art. In a particular embodiment, the light chain is a human light chain.

[0074] The terms "VL" and "VL domain" are used interchangeably and refer to the variable region of an antibody's light chain.

[0075] The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of the heavy chain of an antibody.

[0076] As used herein, the terms “constant region” or “constant domain” are interchangeable and have their ordinary meanings in the art. The constant domain is an antibody portion, and is a carboxy-terminal portion of a light chain and / or heavy chain that is not directly involved in the binding of the antibody to the antigen, for example, but can exhibit various effector functions, such as interactions with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0077] "Fc region (fragment crystallizable region)," "Fc domain," or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the authentic complement system. Thus, the Fc region includes the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. In the case of IgG, the Fc region includes the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of the immunoglobulin heavy chain vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position C226 or P230 (or an amino acid between these two) to the carboxy-terminus of the heavy chain, where the numbering follows the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340, and the CH3 domain is located on the C-terminal side of the Cm domain in the Fc region, i.e., it extends from approximately amino acid 341 to approximately amino acid 447 of IgG. The Fc region may be a natural sequence Fc containing any allotype variant, or a variant Fc (e.g., a non-naturally occurring Fc). Additionally, Fc refers to a region in a separated state, or a region associated with an Fc-containing protein polypeptide, such as a "binding protein containing an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0078] "Hinge," "hinge domain," "hinge region," or "antibody hinge region" refers to a domain of the heavy chain constant region that connects the CH1 domain to the CH2 domain and includes the upper, middle, and lower portions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hinge changes the level of flexibility between the binding domain and the effector domain of the antibody and also provides a site for intermolecular disulfide bonding between two heavy chain constant regions. The sequences of wild-type IgG1, IgG2, IgG3, and IgG4 hinges are known in the art (e.g., Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Vidarsson G. et al., Front Immunol. See 5:520 (released online on October 20, 2014).

[0079] As used herein, "isotype" refers to a type of antibody encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0080] "Allotype" refers to a naturally occurring variant within a specific isotype group that differs by a few amino acids (e.g., Jefferis et al. (2009) mAbs (See 1:1). The antibodies presented herein may have any allotype. Allotypes of IgG1, IgG2, IgG3, and IgG4 are known in the art (e.g., Kabat EA et al., (1991) (ibid.); Vidarsson G. et al., Front Immunol . 5:520 (released online on October 20, 2014); and Lefranc MP, mAbs See 1:4, 1-7(2009)).

[0081] The terms "antibody recognizing an antigen" and "antibody specific to an antigen" are used interchangeably with the term "antibody specifically binding to an antigen" in this specification.

[0082] As used herein, “isolated antibody” refers to an antibody that has substantially no other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to c-Kit has substantially no antibodies that specifically bind to antigens other than c-Kit). However, an isolated antibody that specifically binds to an epitope of c-Kit may have cross-reactivity to other c-Kit proteins from different species.

[0083] "Binding affinity" generally refers to the total sum strength of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise noted, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for partner Y can generally be indicated by a dissociation constant (KD). Affinity may be measured and / or indicated in a number of ways known in the art, including, but not limited to, an equilibrium dissociation constant (KD) and an equilibrium binding constant (KA). KD is calculated from the quotient of koff / kon and is expressed as a molar concentration (M), and KA is calculated from the quotient of kon / koff. kon refers, for example, to the binding rate constant of the antibody to the antigen, and koff refers, for example, to the dissociation rate constant of the antibody to the antigen. kon and koff are for immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIACORE ® or epidemiological exclusion analysis (KinExA ® It can be determined by technology known to those skilled in the art, such as ).

[0084] As used herein, the terms “specifically bind,” “specifically recognize,” “specific binding,” “selective binding,” and “selectively bind” are similar terms in relation to antibodies and refer to molecules (e.g., antibodies) that bind to antigens (e.g., epitopes or immune complexes), and such binding is understood by those skilled in the art. For example, molecules that specifically bind to antigens are, for example, immunoassays, BIACORE ® , KinExA ® When determined by the 3000 instrument (Sapidyne Instruments, Boise, ID) or other analyses known in the art, it can generally bind to other peptides or polypeptides with lower affinity.

[0085] Antibodies are typically 10 -5 to 10 -11 It specifically binds to cognate antigens with high affinity, reflected by a dissociation constant of M or less. Approximately 10 -4 KD exceeding M is generally considered to indicate non-specific binding. Antibodies that "specifically bind" to an antigen refer to antibodies that bind to the antigen and substantially the same antigen with high affinity; this is exemplified, for instance, in immunoassays (e.g., ELISA) or biocores using a defined antigen. ® When determined by surface plasmon resonance (SPR) technology on a 2000 instrument, 10 -7 M or less, preferably 10 -8 M or less, more preferably 10 -9 M or less, more preferably 10 -10 M or less or 10 -11 This means having a KD of M or less, which does not bind with high affinity to unrelated antigens.

[0086] As used herein, the term “antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be a c-Kit or a fragment thereof.

[0087] As used herein, “epitope” is a term in the art referring to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope), or an epitope may be, for example, a polypeptide or a combination of two or more non-contiguous regions of polypeptides (stereotypical, non-linear, discontinuous, or non-contiguous epitope). Epitopes formed from contiguous amino acids are typically retained upon exposure to a denaturing solvent, though not always, and epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. The epitope typically comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acids in a unique spatial conformation. Methods for determining whether an epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art, which include, for example, immunoblotting and immunoprecipitation analysis, in which an overlapping or consecutive peptide (e.g., c-Kit domains 2 and 3) is tested for reactivity with a given antibody (e.g., anti-c-Kit antibody). Methods for determining the spatial stereomorphism of an epitope include those in the art and those disclosed herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0088] With respect to two or more antibodies, the term “binds to the same epitope” means that the antibodies bind to the same segment of an amino acid residue when determined by a given method. Techniques for determining whether antibodies bind to the “same epitope on the c-Kit” as the antibody presented herein include, for example, epitope mapping methods, such as X-ray analysis of the determination of an antigen:antibody complex providing atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of the antibody to an antigen fragment or a mutated variant of the antigen, where the loss of binding due to modifications of amino acid residues within the antigen sequence is considered primarily as an indication of the epitope component. In addition, computer combination methods for epitope mapping may also be used. These methods rely on the ability of the antibody of interest to affinity-separate a specific short peptide from a combination phage display peptide library. Antibodies with the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0089] An antibody that “cross-competes with another antibody for binding to a target” refers to an antibody that inhibits (partially or completely) the binding of the remaining antibody to the target. Whether two antibodies compete with each other for binding to a target—that is, whether one antibody inhibits the binding of the remaining antibody to the target—and the degree of inhibition can be determined using known competition experiments. In certain embodiments, the antibody competes with the other antibody for binding to the target and inhibits this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on whether the antibody is a “blocking antibody” (i.e., a cold antibody that was incubated with the target first). Competition analysis is described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protocol; 2006; This can be done as described in doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999). Competing antibodies bind to the same epitope, overlapping epitope, or adjacent epitope (e.g., by steric hindrance).

[0090] The term "reference antibody" or "reference antibody" refers to an antibody that serves as a standard for the analysis of a competing antibody that binds to the same epitope, an overlapping epitope, or an adjacent epitope, and cross-competes with the competing antibody in binding to the epitope.

[0091] Other competitive combination analyses include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), and sandwich competitive analysis (Stahli et al. Methods in EnzymologySee 9:242 (1983); solid-phase direct biotin-avidin EIA (Kirkland et al., J. Immunol. See 137:3614 (1986); solid-phase direct labeling analysis, solid-phase direct labeling sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 label (Morel et al., Mol. Immunol See . 25(1):7 (1988); solid-phase direct biotin-avidin EIA (Cheung et al., Virology See 176:546 (1990); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. Includes 32:77 (1990) reference).

[0092] c-Kit consists of five extracellular domains comprising IgG-like repeats, a juxtamembrane, and cytoplasmic kinase domains containing ATP-binding (TK1) and phosphotransferase (TK2) domains cleaved by kinase inserts. Upon ligand binding, c-Kit is activated by autophosphorylation via ligand-mediated dimerization. Consequently, signaling mediators including Src, PI3K, STAT1, and JAK2 are recruited to regulate various cell-specific responses such as proliferation, survival, and motility. Furthermore, the overexpression of RTKs is known to be spontaneously activated to induce cell growth in a ligand-independent manner. Overexpression and activation of c-Kit have been reported in various cancers, including GBM, astrocytoma, germ cell cancer, and SCLC, and are correlated with poor prognosis.

[0093] The 2G4 antibody is a fully human anti-c-Kit antibody that exerts a c-Kit inhibitory effect by binding to domain 2 / 3 of c-Kit and blocking SCF binding. The specific c-Kit binding site of 2G4 has been identified using a 2G4 / c-Kit complex docking model (J.-O. Kim et al., International Journal of Biological Macromolecules 159 (2020) 66-78), the above literature is incorporated herein by reference.

[0094] To understand the molecular mechanism of the 2G4 antibody, molecular docking analysis was performed on the crystal structures of 2G4 Fab and c-Kit (PDB ID: 2E9W) using the HADDOCK / ZDOCK program. Residues 26-309 of c-Kit (D1-D3 region, SEQ ID 12) were used in the docking calculation as follows.

[0095] First, blind docking calculations were performed using ZDOCK. The 10 model structures with the highest ZDOCK scores generally indicate that the CDR region of the 2G4 Fab interacts with the D2 and D3 regions (D113-D309; SEQ ID NO. 11) of the c-Kit. Based on the model structures of the 2G4 Fab:c-Kit complex, five residues (R122, Y125, R181, K203, and K205) were selected from the c-Kit D2 region (SEQ ID NO. 9), and five residues (S240, S241, Y243, N260, and W262) were selected from the c-Kit D3 region (SEQ ID NO. 10). The selected residues were expected to be responsible for 2G4 binding. Next, 10 mutant c-Kit proteins were generated, and the binding affinity of 2G4 was compared with that of wild-type and mutant c-Kit proteins using ELISA (Figs. 14a to 14c). The ELISA results showed that the R122, Y125, R181, K203, K205, S261, and H263 residues of c-Kit were important for binding with 2G4.

[0096] The second docking calculation was performed using HADDOCK. The major residues of c-Kit identified by mutagenicity and ELISA analysis, and all residues constituting the CDR of 2G4, were used as constraints. The docking model calculated by HADDOCK showed that the relative orientation and binding interface between 2G4 and c-Kit (i.e., the CDR loop of 2G4 and the D2 / D3 region of c-Kit) were similar to the model calculated by ZDOCK. In the structural model of the 2G4 Fab:c-Kit complex with the highest score, it was confirmed that the heavy chain of 2G4 and the D2-D3 region of c-Kit are closely associated (Fig. 14d). Specifically, the binding interface between 2G4 Fab and c-Kit is formed mainly by electrostatic interactions between the heavy chain CDR of 2G4 and the D2 region of c-Kit. Basic amino acids of c-Kit, including R122, R181, K203, and R205, were found to interact with negatively charged residues of 2G4 (D74H, E119H, D120H, E123H, and D126H) (Fig. 14e). Additional interactions between S261 and H263 of the D3 region of c-Kit and light chain residues of 2G4 (D82L, Q118L, T119L) also contributed to complex formation (Fig. 14e).

[0097] Through the above docking calculation, it was proven that 2G4 is coupled to R122, Y125, R181, K203, R205, S261, and H263 in the D2 / 3 region of the c-Kit.

[0098] Accordingly, the present invention provides an antibody-drug conjugate in which the antibody or its antigen-binding fragment specifically binds to c-Kit. In a specific embodiment of the antibody-drug conjugate, the antibody or its antigen-binding fragment specifically binds to the epitopes of domains 1-3 of human c-Kit (SEQ No. 12), more specifically domains 2 / 3 of human c-Kit (SEQ No. 11), even more specifically the R122 to R205 regions of domain 2 of human c-Kit (SEQ No. 9) and the S240 to H263 regions of domain 3 (SEQ No. 10), most specifically R122, Y125, R181, K203, R205, S261, and H263 of domains 2 / 3 of human c-Kit.

[0099] As used herein, the term “monoclonal antibody” refers to an antibody that exhibits single-link specificity and affinity for a specific epitope, or a composition of antibodies in which all antibodies exhibit single-link specificity and affinity for a specific epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody or antibody composition that exhibits single-link specificity and has a variable and selective constant region derived from a human dummy line immunoglobulin sequence. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic mouse having a genome containing human heavy chain transgene and light chain transgene fused to a transgenic non-human animal, e.g., an immortalized cell.

[0100] The term “recombinant human antibody” comprises all human antibodies produced, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to human immunoglobulin genes or hybridomas produced therefrom; (b) antibodies isolated from host cells transformed to express antibodies, e.g., transfectomas; (c) antibodies isolated from a library of recombinant, recombinant human antibodies; and (d) antibodies produced, expressed, generated, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies include variable and invariant regions utilizing specific human dotline immunoglobulin sequences that are encoded by dotline genes but include subsequent rearrangements and mutations occurring, e.g., during antibody maturation. As known in the art (e.g., Lonberg (2005) Nature Biotech (See . 23(9): 1117-1125), the variable region contains an antigen-binding domain encoded by various genes that have been rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (somatic mutations or supermutations) to increase the affinity of the antibody for the foreign antigen. The constant region will change upon further response to the antigen (i.e., isotype switching). Thus, rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecule, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0101] "Human" antibody (HuMAb) refers to an antibody having a variable region in which both the framework and the CDR region are derived from a human dummy line immunoglobulin sequence. Additionally, if this antibody contains a constant region, the constant region is also derived from a human dummy line immunoglobulin sequence. The antibodies presented herein may contain amino acid residues not encoded by a human dummy line immunoglobulin sequence (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from the dummy line of another mammalian species, such as a mouse, is grafted onto a human framework sequence. The terms "human" antibody and "complete human" antibody are used synonymously.

[0102] The term "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. The addition, deletion, insertion, substitution, or modification of amino acids is permitted as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies possess antigen specificity similar to that of the original antibody.

[0103] "Chimeric antibodies" refer to antibodies in which a variable region is derived from one species and a constant region is derived from another species, for example, an antibody in which a variable region is derived from a mouse antibody and a constant region is derived from a human antibody.

[0104] As used herein, the term “cross-reactive” refers to the ability of the antibodies presented herein to bind to c-Kit from different species. For example, the antibodies presented herein that bind to human c-Kit may also bind to c-Kit of other species (e.g., mouse c-Kit). Such cross-reactivity may be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or by detecting binding with cells physiologically expressing c-Kit, or by detecting functional interactions. Methods for determining cross-reactivity include standard binding assays disclosed herein, e.g., BIACORE ® BIACORE using the 2000 SPR machine (Biacore AB, Uppsala, Sweden) ® Includes surface plasmon resonance (SPR) analysis or flow cytometry techniques.

[0105] The term "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having a basic side chain (e.g., lysine, arginine, histidine), an acidic side chain (e.g., aspartic acid, glutamic acid), a non-polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), a non-polar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), a beta-branched side chain (e.g., threonine, valine, isoleucine), and an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, the expected non-essential amino acid residue in the anti-c-Kit antibody is substituted with another amino acid residue from the same side chain family. Methods for identifying nucleotide and amino acid-conserving substitutions that do not impair antigen binding are well known in the art (e.g., Brummell et al., Biochem . 32: 1180-1187 (1993); Kobayashi et al. Protein Eng . 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. See USA 94:412-417 (1997).

[0106] The term “substantial homology” indicates that two polypeptides, or their specified sequences, are identical when optimally aligned and compared, having appropriate amino acid insertions or deletions in at least about 80%, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids.

[0107] The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two sequences (i.e., homology % = number of identical positions / total number of positions x 100). The comparison of sequences and the percentage of identity between two sequences can be determined using mathematical algorithms such as those described in the non-limiting examples below.

[0108] The percentage of identity between two amino acid sequences is Needleman and Wunsch (integrated into the GAP program of the GCG software package) J. Mol. Biol. (48):444-453 (1970)) can be determined using the algorithm (available at http: / / www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6.

[0109] The nucleic acid and protein sequences presented in this specification may be used, for example, as "query sequences" to perform a search in public databases to identify related sequences. Such a search may be performed using the literature (Altschul, et al. (1990) J. Mol. Biol. This can be performed using the NBLAST and XBLAST programs (version 2.0) of 215:403-10). BLAST nucleotide search can be performed using the NBLAST program, score = 100, word length = 12, thereby obtaining nucleotide sequences homologous to the nucleic acid molecules presented herein. BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3, thereby obtaining amino acid sequences homologous to the protein molecules presented herein. To obtain gap alignment for comparison purposes, Gapped BLAST is used in the literature (Altschul et al., (1997) Nucleic Acids Res It may be used as described in . 25(17): 3389-3402). When using BLAST and Gapped BLAST programs, the default variables of each program (e.g., XBLAST and NBLAST) may be used (see worldwideweb.ncbi.nlm.nih.gov).

[0110] As used herein, the term “linked” refers to the association of two or more molecules. The linkage may be a covalent linkage or a non-covalent linkage. The linkage may also be a genetic linkage (i.e., recombinantly fused). Such linkage may be achieved using various techniques recognized in the art, such as chemical conjugation and the generation of recombinant proteins.

[0111] As used herein, the term “antibody-drug conjugate” refers to the connection between an antibody or its antigen-binding fragment and another agent, such as an anticancer agent, chemotherapy agent, toxin, immunotherapy agent, imaging probe, spectroscopic probe, etc. Such connection may be a covalent bond or a non-covalent interaction, such as an interaction through electrostatic force. Various linkers known in the art may be used to form antibody-drug conjugates. Additionally, antibody-drug conjugates may be provided in the form of fusion proteins that can be expressed from polynucleotides encoding antibody-drug conjugates. As used herein, “fusion protein” refers to a protein produced through the linkage of two or more genes or gene fragments that originally code for distinct proteins (including peptides and polypeptides). Translation of the fusion genes results in a single protein with functional properties derived from each of the original proteins.

[0112] In this specification, "spacer" refers to a substance that prevents the aggregation of antibody-drug conjugates in the bloodstream and provides stability in the bloodstream and desired pharmacokinetic properties.

[0113] As used herein, the term “linker” is any chemical moiety capable of linking an antibody, an antibody fragment (e.g., an antigen-binding fragment), or a functional equivalent to another moiety, e.g., a drug moiety. Under conditions where the compound or antibody is still active, the linker may be susceptible to cleavage, e.g., acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterarase-induced cleavage, and disulfide bond cleavage (cleavable linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker). In some embodiments, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0114] "Non-cleavable linkers" possess relatively high plasma stability and are resistant to proteolysis. After introduction into the cell, the antibody must be cleaved for the drug to be released in the form of a complex with the linker, and this complex possesses drug activity. Thioether linkers are representative non-cleavable linkers that allow the drug to detach and be released after the antibody is non-selectively cleaved within the cell. Kadcyla ® ) adopts a non-cutting method.

[0115] A "cleavable linker" refers to a linker that can be cleaved in response to specific environmental factors to release a drug into the cytoplasm. Cleaving types include enzymatic and non-enzymatic types.

[0116] Enzymatic cleavage forms are cleaved by enzymes such as cathepsin B, β-glucuronidase, phosphatase, pyrophosphatase, and sulfatase. Peptide linkers are degraded by proteases, and their plasma stability is high due to the presence of protease inhibitors in plasma. Cathepsin B is a representative protease used in antibody-drug conjugates, and because it is present at high levels in tumor tissues, it imparts tumor selectivity to antibody-drug conjugates. Peptide linkers are typically dipeptides formed by the conjugation of two amino acids. β-glucuronide linkers are degraded by β-glucuronidase, a glycolytic enzyme present in lysosomes. β-glucuronidase is overexpressed in some tumor cells, conferring tumor specificity.

[0117] Non-enzymatic cleavage types include acid-labile linkers and oxidation-reduction reaction linkers.

[0118] Acid-unstable linkers are linkers with relatively low stability that were developed in the early days, but they are still in use. A representative example is the hydrazone linker, which is internalized into tumor cells and then hydrolyzed in the weakly acidic environment of endosomes and lysosomes to release drugs.

[0119] Disulfide linkers are representative redox linkers. After internalization, the linker is degraded by reducing agents such as disulfide exchange or glutathione, releasing cytotoxic drugs. Glutathione is about 1,000 times more concentrated in hypoxic tumor cells than in normal cells, thereby conferring tumor cell selectivity to ADCs.

[0120] Procharged linkers are derived from charged crosslinking reagents that retain their charge after incorporation into antibody-drug conjugates. An example of a procharged linker can be found in U.S. Patent Publication No. 2009 / 0274713.

[0121] II. Antibody-Drug Conjugate (ADC)

[0122] According to one aspect of the present invention, the present invention provides an antibody-drug conjugate of the following formula 1:

[0123] <Chemical Formula 1>

[0124] Ab-(L) x -(D) y

[0125] In the above formula,

[0126] Ab is an anti-c-Kit antibody or its antigen-binding fragment that specifically binds to the epitope of human c-Kit at SEQ ID NOs 9 and 10;

[0127] L is a linker including a cleavable linker;

[0128] D is a drug moiety;

[0129] x is an integer from 1 to 8;

[0130] y is an integer from 1 to 8.

[0131] In some embodiments, the Ab is an antibody or its antigen-binding fragment that specifically binds to one or more of R122, Y125, R181, K203, R205, S261, and H263 in SEQ ID NO. 12. Preferably, the Ab is an antibody or its antigen-binding fragment that specifically binds to R122, Y125, R181, K203, R205, S261, and H263 in SEQ ID NO. 12. In this case, SEQ ID NO. 12 is the sequence (Q26 to D309) of domains 1 to 3 of a human c-Kit excluding 25 signal peptides, and the number of each amino acid is the number including 25 signal peptides. Accordingly, for example, R122 means the 97th amino acid sequence in SEQ ID NO. 12, Y125 means the 100th amino acid sequence, and the remaining amino acids can be interpreted in the same way.

[0132] In some embodiments, the antibody or its antigen-binding fragment cross-competes for binding to the human c-Kit epitope in SEQ ID NO. 9 and SEQ ID NO. 10 with a reference antibody comprising the heavy chain CDR1 of SEQ ID NO. 1, the heavy chain CDR2 of SEQ ID NO. 2, the heavy chain CDR3 of SEQ ID NO. 3, the light chain CDR1 of SEQ ID NO. 4, the light chain CDR2 of SEQ ID NO. 5, and the light chain CDR3 of SEQ ID NO. 6.

[0133] In some embodiments, the antibody or its antigen-binding fragment cross-competes with a reference antibody comprising a heavy chain variable domain represented by SEQ ID NO. 7 and a light chain variable domain represented by SEQ ID NO. 8 for binding to the epitope of human c-Kit at SEQ ID NO. 9 and SEQ ID NO. 10.

[0134] In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain CDR1 of SEQ ID NO. 1, the heavy chain CDR2 of SEQ ID NO. 2, the heavy chain CDR3 of SEQ ID NO. 3, the light chain CDR1 of SEQ ID NO. 4, the light chain CDR2 of SEQ ID NO. 5, and the light chain CDR3 of SEQ ID NO. 6.

[0135] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable domain represented by SEQ ID NO. 7 and a light chain variable domain represented by SEQ ID NO. 8.

[0136] The above antibody or its antigen-binding fragment may have one or two amino acids in the CDR modified, deleted, or substituted, but is not limited to this.

[0137] In some embodiments, the antibody or its antigen-binding fragment may maintain 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more of identity across the heavy chain variable or light chain heavy chain regions.

[0138] In some embodiments, the antibody or its antigen-binding fragment may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single-chain antibody (scFv), or an antibody fragment.

[0139] In some embodiments, the antibody or its antigen-binding fragment has an equilibrium dissociation constant K for human c-KIT. D The value is 10 -11 M or less, specifically, for example, approximately 2.8237 (±0.9) X 10⁻⁶ -12 It could be M.

[0140] The above-mentioned cleavable linker includes, but is not limited to, a linker selected from the group consisting of valine-citrulline-p-aminobenzyl carbamoyl (Val-Cit-PAB), alanine-phenylalanine-p-aminobenzyl carbamoyl (Ala-Phe-PAB), alanine-alanine-p-aminobenzyl carbamoyl (Ala-Ala-PAB), valine-alanine-p-aminobenzyl carbamoyl (Val-Ala-PAB), phenylalanine-lysine-p-aminobenzyl carbamoyl (Phe-Lys-PAB), alanine-alanine-glycine-p-aminobenzyl carbamoyl (Ala-Ala-Gly-PAB) and glycine-glycine-glycine-p-aminobenzyl carbamoyl (Gly-Gly-Gly-PAB) linkers.

[0141] The above L may include a spacer in a form to which a polymer is bonded or not bonded.

[0142] According to one embodiment of the present invention, the L is , , (for example, ), , , , and It is a linker comprising a linker derived from a crosslinking reagent comprising a substituent selected from the group consisting of (for example, When prepared using a crosslinking reagent containing a substituent of ), for example, the linker is a thiobridge ® It may include a disulfide bridge connecting two cysteine ​​residues, as in the Abzena method.

[0143] In the above chemical formula, the wavy line ( ) indicates the site where another linker or S or D binds.

[0144] The above R may be hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, cyano, nitro, phenyl, toluene, or halophenyl.

[0145] In some embodiments, L is a linker comprising a spacer in which the polymer is bonded or is not bonded.

[0146] In some embodiments, the above L comprises the structure of the following chemical formula 2:

[0147] <Chemical Formula 2>

[0148] (L1) m -(S) n -(L2) p

[0149] In the above formula,

[0150] L1 is a linker connecting the above-mentioned Ab and S or Ab and L2;

[0151] S is a spacer in a form where the polymer is bonded or not bonded;

[0152] L2 is a cleavable linker;

[0153] m is an integer from 0 to 8;

[0154] n is an integer from 0 to 8;

[0155] p is an integer from 1 to 8.

[0156] In some embodiments, m is 1 to 4, n is 1 to 4, and p is 1 to 4.

[0157] In some embodiments, L1 comprises a linker selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker, and a dicarboxylic acid-based linker.

[0158] In some embodiments, L or L1 is , , , , , , , , , , , , , , , , , , , , , , , , , , , , or Includes

[0159] In the above chemical formula, the wavy line ( ) indicates the site where Ab, or S, L2, or D binds.

[0160] In some embodiments, the above L1 is , , (for example, ), , , , and It is a linker comprising a linker derived from a crosslinking reagent containing a substituent selected from the group consisting of

[0161] In the above chemical formula, the wavy line ( ) indicates the site where S binds.

[0162] The above R may be hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, cyano, nitro, phenyl, toluene, or halophenyl.

[0163] According to one embodiment of the present invention, the S (spacer) is , , , and It can be selected from a group consisting of

[0164] In the above chemical formula, the wavy line ( ) indicates the site where L1 or L2 is joined, preferably L1 is joined to the left wavy line side and L2 is joined to the right wavy line side.

[0165] In some embodiments, the above S may be a spacer comprising aspartate, glutamate, or a combination thereof in a form to which the polymer is bonded or not bonded.

[0166] Examples of the above polymers include polyalkylene, polyalkylene glycol, polyvinylpyrrolidone, polyacrylate, polyoxazoline, polyvinyl alcohol, polyacrylamide or polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(orthoester), polycarbonate, poly(iminocarbonate), polyamide, copolymer of divinyl ether-maleic anhydride or styrene-maleic anhydride, polysaccharide, or polyglutamic acid, but are not limited thereto.

[0167] In some embodiments, the polymer is polyethylene or polyethylene glycol.

[0168] The above polyethylene glycol may contain 10 to 30 ethylene glycol units (-O-CH2-CH2-), for example, 24 units.

[0169] According to one embodiment of the present invention, L comprises a linker selected from the group consisting of the following chemical formulas 3 to 5:

[0170] <Chemical Formula 3>

[0171]

[0172] ,

[0173] <Chemical Formula 4>

[0174] or

[0175] <Chemical Formula 5>

[0176] .

[0177] In the above chemical formula, the wavy line ( ) indicates the site where Ab or D binds, preferably Ab binds to the left wavy line and D binds to the right wavy line.

[0178] The above D is preferably a drug moiety selected from the group consisting of V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule inhibitors, auristatin, dolastatin, methansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear efflux of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA insertants, DNA small groove binders, and DHFR inhibitors.

[0179] An antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the present disclosure may be conjugated to a drug moiety that modifies a desired biological response. The drug moiety should not be interpreted as being limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide possessing the desired biological activity. Such proteins may include, for example, toxins such as abrine, lysine A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, cytokine, apoptosis agent, anti-angiogenic agent, or biological response modifier, for example, lymphokine.

[0180] In some embodiments, an antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent of the present disclosure is conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Examples of cytotoxins include taxanes (e.g., see International (PCT) Patent Application No. WO 01 / 38318 and PCT / US03 / 02675), DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulisin analogs, duocarmycin analogs, auristatin E, auristatin F, meitansinoids, and cytotoxic agents comprising a reactive polyethylene glycol moiety (e.g., see literature [Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000)], [Suzawa et al., Bioorg. Med. Chem. , 8, 2175-84 (2000)], [Ichimura et al., J. Antibiot . (Tokyo), 44, 1045-53 (1991)], [Francisco et al., Blood[2003 15;102(4):1458-65], U.S. Patent Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001 / 0036923 A1, pending U.S. Patent Application Serial Nos. 10 / 024,290 and 10 / 116,053, and International (PCT) Patent Application No. WO 01 / 49698), Taxone, Cytocalcin B, Gramicidin D, Ethidium Bromide, Emetine, Mitomycin, Etoposide, Tenofoside, Vincristine, Vinblastine, T. Colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof, but are not limited thereto. Therapeutic agents are, for example, anti-metabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepah, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and antramycin (AMC)), and anti-mitotic agents (e.g., It also includes vincristine and vinblastine (see, for example, Seattle Genetics US20090304721).

[0181] Other examples of cytotoxins that may be conjugated to the antibodies, antibody fragments (antigen-binding fragments), or functional equivalents of the present disclosure include duocarmycin, calikiamycin, meitansin, and auristatin, and derivatives thereof.

[0182] Methods for conjugating various types of cytotoxins, linkers, and therapeutic agents to antibodies are known in the relevant field, for example, in the literature [Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215]; [Trail et al., (2003) Cancer Immunol. Immunother. 52:328-337]; [Payne, (2003) Cancer Cell 3:207-212]; [Allen, (2002) Nat. Rev. Cancer 2:750-763]; [Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091]; [Senter and Springer, (2001) Adv. Drug Deliv. Rev. Refer to 53:247-264.

[0183] Antibodies, antibody fragments (e.g., antigen-binding fragments), or functional equivalents of the present disclosure may be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals referred to as radioisotopes. Examples of radioisotopes that may be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for producing radioisotopes are established in the art. Examples of radioisotopes, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), are commercially available, and similar methods may be used to produce radioisotopes using antibodies disclosed herein. In a specific respect, the macrocyclic chelator is 1,4,7,10-tetraazcyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to an antibody via a linker molecule. Such linker molecules are commonly known in the relevant art, and the literature [Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90]; [Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7]; and [Zimmerman et al., (1999) Nucl. Med. Biol. It is described in [26(8):943-50] (each full text included for reference).

[0184] In some embodiments, the above D is a microtubule inhibitor (MTI) moiety.

[0185] The above microtubule inhibitor is not limited thereto, but preferably SN-38 ((4S)-4,11-Diethyl-4,9-dihydroxy-1,4-dihydro-3 H ,14 H-pyrano[3′,4′:6,7]indolizino[1,2- b It may include a drug selected from the group consisting of quinoline-3,14-dione), auristatin, dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethyldolastatin 10 (MMAD), or a combination thereof.

[0186] SN-38 inhibits DNA topoisomerase I, thereby preventing cell division. It has lower efficacy compared to existing drugs, resulting in fewer side effects, and can be used to increase the efficacy relative to side effects of antibody-drug conjugates by increasing DAR.

[0187] The antibody or its antigen-binding fragment preferably specifically binds to an epitope of domain 2 / 3 of the human c-Kit represented by SEQ ID NO. 11 (i.e., D113 to D309), more preferably specifically binds to an epitope of domain 2 of the human c-Kit represented by SEQ ID NO. 9 (i.e., R122 to R205) and / or an epitope of domain 3 of the human c-Kit represented by SEQ ID NO. 10 (i.e., S240 to H263), and even more preferably specifically binds to one or more amino acid residues selected from the group consisting of R122, Y125, R181, K203, and R205 in SEQ ID NO. 9 and / or one or more amino acid residues selected from the group consisting of S261 and H263 in SEQ ID NO. 10.

[0188] In some embodiments, the antibody or its antigen-binding fragment cross-competes for binding to the epitope of domain 2 / 3 of the human c-Kit of SEQ ID NO. 11 (i.e., D113 to D309) with a reference antibody comprising a heavy chain variable domain represented by SEQ ID NO. 7 and a light chain variable domain represented by SEQ ID NO. 8.

[0189] According to one embodiment of the present invention, the antibody-drug conjugate is selected from the group consisting of the following chemical formulas 6 to 8:

[0190] <Chemical Formula 6>

[0191] ,

[0192] <Chemical Formula 7>

[0193] and

[0194] <Chemical Formula 8>

[0195] .

[0196] In the above formula, a is an integer from 1 to 8, and b and c are integers from 1 to 4, respectively.

[0197] According to another aspect of the present invention, the present invention provides an antibody-drug conjugate selected from the group consisting of the following chemical formulas 6 to 8:

[0198] <Chemical Formula 6>

[0199]

[0200] ,

[0201] <Chemical Formula 7>

[0202] and

[0203] <Chemical Formula 8>

[0204] .

[0205] In the above formula,

[0206] Ab is an antibody or its antigen-binding fragment comprising heavy chain CDR1 of SEQ ID NO. 1, heavy chain CDR2 of SEQ ID NO. 2, heavy chain CDR3 of SEQ ID NO. 3, light chain CDR1 of SEQ ID NO. 4, light chain CDR2 of SEQ ID NO. 5, and light chain CDR3 of SEQ ID NO. 6;

[0207] a is an integer from 1 to 8, and b and c are integers from 1 to 4, respectively.

[0208] In some embodiments, the antibody-drug conjugate has the structure of Formula 7, where b is 2.

[0209] In some embodiments, the antibody-drug conjugate has the structure of Formula 7, where b is 4.

[0210] III. Composition

[0211] According to another aspect of the present invention, the present invention provides a composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0212] In some embodiments, the composition is a pharmaceutical composition for the prevention or treatment of cancer.

[0213] According to another aspect of the present invention, the present invention provides a therapeutic use of a composition.

[0214] In some specific examples, the above use is for the treatment of cancer.

[0215] According to another aspect of the present invention, the present invention provides a use for the preparation of the above pharmaceutical composition.

[0216] According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising the step of administering the composition to a subject in need thereof.

[0217] A method for treating a tumor (or cancer) of a subject is disclosed herein, comprising administering the antibody-drug conjugate or a pharmaceutically acceptable salt thereof to the subject. In certain embodiments, the antibody-drug conjugate specifically binds to the c-Kit protein and reduces c-Kit activity.

[0218] In some embodiments, the method disclosed herein inhibits and / or reduces tumor growth of a subject. In certain embodiments, tumor growth (e.g., tumor volume or weight) is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a reference (e.g., the corresponding tumor volume or weight in a subject that has not received the composition disclosed herein, e.g., the antibody-drug conjugate). In some embodiments, the method disclosed herein increases mean tumor growth inhibition (TGI) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a reference (e.g., a corresponding frequency in subjects who did not receive the composition disclosed herein, e.g., the antibody-drug conjugate). In some embodiments, administration of the composition disclosed herein (e.g., the antibody-drug conjugate) increases mean survival by at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or more compared to a reference (e.g., a corresponding value in subjects who did not receive the composition disclosed herein, e.g., the antibody-drug conjugate).

[0219] In some embodiments, the tumors that can be treated by the method disclosed herein are typically derived from cancers that are responsive to conventional anticancer agents and cancers that are typically non-responsive to conventional anticancer agents. In some embodiments, the cancer is a solid tumor or a cancer having a blood malignancy (liquid tumor).

[0220] Non-limiting examples of cancers requiring treatment include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-resistant prostatic adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colorectal carcinoma, and head and neck cancer (or carcinoma), germ cell tumors, pediatric sarcoma, nasal natural killer, melanoma (e.g., metastatic malignant melanoma, e.g., cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, esophageal cancer, small intestine cancer, colorectal cancer. Mast cell tumors, cancers of the endocrine system, cancers of the parathyroid glands, cancers of the adrenal glands, soft tissue sarcomas, cancers of the urethra, cancers of the genitals, solid tumors of children, cancers of the ureters, carcinomas of the renal pelvis, neovascularization, pituitary adenoma, Kaposi's sarcoma, epithelial carcinomas, squamous cell carcinomas, T-cell lymphomas, environmentally induced cancers including those caused by asbestos, virus-associated cancers or cancers of viral origin (e.g., human papillomavirus (HPV-associated or --origin tumors)), and hematological malignancies derived from either of the two major blood cell lines, namely, myeloid cell lines (which produce granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (which produce B, T, NK, and plasma cells), e.g., all types of leukemia, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myeloid leukemias, e.g., acute leukemia (ALL), acute myeloid Leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloid leukemia (M1), myeloid leukemia (M2;(with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or eosinophilic M4 variant [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; Lymphoma, e.g., Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoblastoma lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, centrioles lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histocytic lymphoma vera, primary efflux lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of the lymphatic system, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sizzari syndrome), and lymphoblastic lymphoma with Waldenström macroglobulinemia (LPL); Myeloma, e.g. IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called painless myeloma), solitary plasmocytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), morphogenetic cell lymphoma; hematopoietic tumors of the myeloid lineage, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; seminomas, teratocarcinomas, and tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma;and other tumors including melanoma, xeroderma pigmentosum, acanthoma keratoceti, seminoma, thyroid follicular carcinoma and teratocarcinoma; hematopoietic tumors of the lymphatic system, including, for example but not limited to, small cell and cerebral-like cell types, T-cell and T-cell tumors including T-cell disorders such as T-prolymphocytic leukemia (T-PLL); T-cell type macrogranulocytic leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); vascular centrosome (nasal) T-cell lymphoma; cancer of the head or neck, kidney cancer, rectal cancer, cancer of the thyroid; acute myeloid lymphoma, and any combination thereof.;

[0221] According to one embodiment of the present invention, the cancer is one or more cancers selected from the group consisting of esophageal cancer, gastric cancer, GIST, colorectal cancer, rectal cancer, lung cancer, in particular SCLC, breast cancer, mast cell tumor and leukemia, in particular AML.

[0222] In some embodiments, the method disclosed herein may also be used to treat metastatic cancer, unresectable, refractory cancer (e.g., cancer resistant to conventional cancer therapies, e.g., immunotherapy, e.g., therapy with blocking PD-(L)1 antibodies), and / or recurrent cancer. In certain embodiments, the method disclosed herein may be used to treat recurrent cancer.

[0223] In some embodiments, the method disclosed herein includes conventional cancer therapy such as chemotherapy. For example, it may be administered in combination with imatinib and other c-KIT pathway inhibitors.

[0224] In some embodiments, the method disclosed herein effectively increases the duration of survival of a subject (e.g., one in which a tumor has invaded). For example, the duration of survival of the subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year or more compared to a reference subject (e.g., another subject not treated with the composition disclosed herein, e.g., said antibody-drug conjugate). In another embodiment, the method disclosed herein increases the duration of survival of a subject to a level longer than the duration of survival of a reference subject (e.g., another subject not treated with the composition disclosed herein, e.g., said antibody-drug conjugate) (about 1 month longer, about 2 months longer, about 3 months longer, about 4 months longer, about 5 months longer, about 6 months longer, about 7 months longer, about 8 months longer, about 9 months longer, about 10 months longer, about 11 months longer, or about 1 year longer).

[0225] In some embodiments, the method of the present invention increases the duration of disease progression-free survival of a subject. For example, the duration of disease progression-free survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 1 year compared to a reference subject (e.g., another subject not treated with the composition disclosed herein, e.g., said antibody-drug conjugate).

[0226] In some embodiments, the method disclosed herein effectively increases the reaction rate in a group of subjects. For example, the reaction rate in a group of subjects is increased by at least about 2%, at least about 3%, at least about 4%, 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 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% compared to a reference subject (e.g., a composition disclosed herein, e.g., another subject not treated with said antibody-drug conjugate).

[0227] In some embodiments, the subject to be treated in the method is a non-human animal, e.g., a rat or a mouse. In some embodiments, the subject to be treated in the method is a human.

[0228] The present invention also includes a method for treating a target cancer in combination with other anticancer agents. In some embodiments, the antibody-drug conjugate useful in the method of the present invention may be administered as a combination therapy, that is, in combination with at least one other anticancer agent and / or immunomodulator, e.g., a T-cell stimulating (e.g., activating) agent. In some embodiments, the antibody-drug conjugate useful in the method of the present invention may be administered in combination with other compounds, drugs, and / or agents used in the treatment of cancer. Such compounds, drugs, and / or agents may include, e.g., chemotherapy drugs, small molecule drugs, or antibodies that stimulate an immune response against cancer. In some embodiments, the method disclosed herein is used in combination with standard treatments (e.g., surgery, radiation, and chemotherapy). In other embodiments, the method disclosed herein is used as a maintenance therapy, e.g., as a therapy intended to prevent the development or recurrence of a tumor.

[0229] In some embodiments, the antibody-drug conjugate disclosed herein may be used in combination with one or more additional cancer agents comprising an immunotherapeutic agent, a chemotherapeutic agent, a targeted therapeutic agent, a radiotherapeutic agent (radiotherapy), or any combination thereof.

[0230] Compositions comprising the antibody-drug conjugate disclosed herein having a desired degree of purity among physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA) are disclosed herein. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dose and concentration used and include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, e.g., TWEEN ® , PLURONICS ® or includes polyethylene glycol (PEG).

[0231] In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate disclosed herein in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of the antibody-drug conjugate disclosed herein, and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the antibody-drug conjugate is the only active ingredient included in the pharmaceutical composition. The pharmaceutical composition disclosed herein may be useful for reducing c-Kit activity and thereby treating cancer.

[0232] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, metal ion blocking or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, and dextrose and lactate Ringer's injection. Non-aqueous vehicles include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers, and these include phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspension and dispersants include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN ®80) includes. The metal ion blocking or chelating agent includes EDTA. The pharmaceutical carrier also includes ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0233] The pharmaceutical composition may be formulated for any route of administration to the subject. Specific examples of routes of administration include intranasal, oral, parenteral, intravertebral, intraventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also considered. The injectable substance may be prepared in conventional forms, as a liquid solution or suspension, as a solid form suitable for becoming a solution or suspension in liquid before injection, or as an emulsion. The injectable substance, solution, and emulsion also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0234] Manufactured products for parenteral administration of antibody-drug conjugates include sterile solutions ready for injection, sterile dry soluble products that can be combined with a solvent immediately before use, such as lyophilized powders, sterile suspensions ready for injection, sterile dry insoluble products that can be combined with a vehicle immediately before use, and sterile emulsions. The solution may be aqueous or non-aqueous.

[0235] The antibody-drug conjugate disclosed herein or its pharmaceutically acceptable salts may also be formulated to target specific tissues, receptors, or other body regions of the target to be treated. Various targeting methods are known to those skilled in the art. The use of such targeting methods in the compositions is considered. For non-limiting examples of targeting methods, see U.S. Patents No. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. In specific embodiments, the antibody-drug conjugate disclosed herein or a pharmaceutically acceptable salt thereof may be used to treat cancer.

[0236] The composition used for in vivo administration can be sterilized. For example, it can be sterilized by filtration through a sterile filter membrane.

[0237] According to another aspect of the present invention, a composition for diagnosing cancer is provided, comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0238] IX. Kit

[0239] A kit comprising one or more antibody-drug conjugates disclosed herein or pharmaceutically acceptable salts thereof is disclosed herein. In some embodiments, a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the composition disclosed herein, comprising one or more antibody-drug conjugates disclosed herein or pharmaceutically acceptable salts thereof, and optional instructions for use is disclosed herein. In some embodiments, the kit comprises the pharmaceutical composition disclosed herein and any prophylactic or therapeutic agent disclosed herein. In some embodiments, the kit comprises a composition disclosed herein and instructions for a method of detection and / or diagnosis of cancer.

[0240] In some embodiments, a method for providing information for the diagnosis of cancer is provided, comprising the step of treating a sample separated from a subject with an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0241] X. Production method

[0242] A method for producing one or more anti-c-Kit antibody drug conjugates disclosed herein is disclosed herein.

[0243] The composite of the present disclosure may be prepared by any method known in the relevant art, such as those described in U.S. Registered Patents No. 7,811,572, 6,411,163, 7,368,565, and 8,163,888, and U.S. Published Patents No. 2011 / 0003969, 2011 / 0166319, 2012 / 0253021, and 2012 / 0259100. The entire teachings of these patents and patent application publications are incorporated herein by reference.

[0244] In addition, various types of cytotoxins, linkers, and methods for conjugating drugs to antibodies are known in the relevant field, for example, in the literature (Saito et al., Adv. Drug Deliv. Rev.55:199-215 (2003); Trail et al., Cancer Immunol. Immunother . 52:328-337(2003); Payne, Cancer Cell 3:207-212(2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer, Adv. Drug Deliv. Rev. Refer to 53:247-264(2001)].

[0245] In some embodiments, the above production method is to an antibody or its antigen-binding fragment (Ab) that specifically binds to a human c-Kit (L1) m -(S) n -(L2) p -(D) q Includes a step of joining:

[0246] In the above formula,

[0247] L1 is a linker connecting the above-mentioned Ab and S or Ab and L2;

[0248] S is a spacer in a form where the polymer is bonded or not bonded;

[0249] L2 is a cleavable linker;

[0250] D is a drug moiety;

[0251] m is an integer from 0 to 8;

[0252] n is an integer from 0 to 8;

[0253] p is an integer from 1 to 8;

[0254] q is an integer from 1 to 8.

[0255] The definitions and related descriptions of the above terms are as described in I. Definitions and II. Antibody-Drug Conjugate (ADC).

[0256] In some embodiments, the production method comprises the step of conjugating a linker (L) and a drug (D) to an antibody or its antigen-binding fragment comprising the heavy chain CDR1 of SEQ ID NO. 1, the heavy chain CDR2 of SEQ ID NO. 2, the heavy chain CDR3 of SEQ ID NO. 3, the light chain CDR1 of SEQ ID NO. 4, the light chain CDR2 of SEQ ID NO. 5, and the light chain CDR3 of SEQ ID NO. 6, wherein L comprises a cleavable linker.

[0257] In some embodiments, the above D is a microtubule inhibitor moiety.

[0258] In some embodiments, m is 1 to 4, n is 1 to 4, p is 1 to 4, and q is 1 to 8. Effects of the invention

[0259] The features and advantages of the present invention are summarized as follows:

[0260] (i) The present invention provides an antibody-drug conjugate comprising an antibody that specifically binds to human c-Kit, a cleavable linker, and a microtubule inhibitor moiety.

[0261] (ii) In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising an antibody-drug conjugate as an active ingredient.

[0262] (iii) The antibody-drug conjugate of the present invention has the characteristic of possessing potent anticancer activity against c-Kit-positive and negative cancer cell lines and / or imatinib-resistant cancer cell lines, thereby inducing complete remission. Brief explanation of the drawing

[0263] FIG. 1 illustrates a method for manufacturing an ADC using a cleavable linker. FIG. 1a shows the linker-drug used in one embodiment, and FIG. 1b shows ThioBridge ® This shows the schematic manufacturing process for -Glu-(Val-Cit-PAB-MMAE)-PEG(24u). Figure 2 shows the results of an in vitro cell viability analysis performed to confirm the efficacy of the ADC of the present invention in c-Kit-positive and c-Kit-negative cancer cell lines. Figure 3 shows the results confirming the in vivo efficacy of the ADC of the present invention against GIST in mice (GIST T1 cells) (Figure 3a shows the results for PoC-DM1; Figure 3b shows 003-1, 2G4 ThioBridge ® Results for -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2; Fig. 3c is 004-2, 2G4 ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) results for DAR 4; Fig. 3d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE). Figure 4 shows the results confirming the in vivo efficacy of the ADC of the present invention against GIST in mice (GIST-430 / 654 cells) (Figure 4a shows the results for PoC-DM1; Figure 4b shows 003-1, 2G4 ThioBridge ® Results for -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2; Fig. 4c is 004-2, 2G4 ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) results for DAR 4; Fig. 4d is the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE; Fig. 4e is 007-1, 2G4-ThioBridge ® -Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 represents the result). Figure 5 shows the results confirming the in vivo efficacy of the ADC of the present invention against SCLC in mice (SCLC-H526 cells) (Figure 5a shows the results for PoC-DM1; Figure 5b shows 003-1, 2G4 ThioBridge ®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2 results; Fig. 5c is 004-2, 2G4 ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) results for DAR 4; Fig. 5d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE). Figure 6 shows the results of confirming the in vivo efficacy of the ADC of the present invention against mast cell tumors in mice (HMC 1.2 cells). Figure 7 shows the results confirming the efficacy of the ADC of the present invention upon tumor re-inoculation. Figure 8 shows the results of confirming the in vivo efficacy of the ADC of the present invention against AML in mice (Kasumi-1 cells). Figure 9 shows the results confirming the in vivo efficacy of the ADC of the present invention against breast cancer in mice (MDA-MB-468 cells) (Figure 9a shows the results for PoC-DM1; Figure 9b shows 003-1, 2G4 ThioBridge ® Results for -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2; Fig. 9c is 004-2, 2G4 ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) results for DAR 4; Fig. 9d shows the results for 003-2, 2G4-MC-Val-Cit-PAB-MMAE). Figure 10 shows the results of a re-challenge analysis of SCLC in mice (SCLC-H526 cells) for the ADC of the present invention. Figure 11 shows the results of a re-challenge analysis of the ADC of the present invention on mast cell tumors in mice (HMC 1.2 cells). Figure 12 shows the results of a pre-toxicity analysis performed in vivo on the ADC of the present invention at a high concentration (10 mg / kg). Figure 13 shows the results of in vivo pre-toxicity analysis performed on the ADC of the present invention at higher concentrations (20 / 40 / 60 mg / kg) (Figure 13a shows the results for the 2G4 antibody; Figure 13b shows the results for PoC-DM1; Figure 13c shows the results for 003-1, 2G4 ThioBridge ® Results for -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2; Fig. 13d is 004-2, 2G4 ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) shows the results for DAR 4). Figure 14 shows the docking model of 2G4 Fab:c-Kit. (Figure 14a) A summary of the c-Kit mutants. (Figures 14b and c) Wild-type and mutant c-Kit proteins were purified and coated onto 96-well plates after serial dilution. Binding of 2G4 was investigated by ELISA. (Figure 14d) Structural model of the 2G4 Fab:c-Kit complex with the highest score calculated by HADDOCK. The heavy chain (H) variable (V) and constant (C) regions and the light chain (L) variable (V) and constant (C) regions of 2G4 are labeled VH, CH1, VL, and CL, respectively. (Figure 14e) Shows the stick representation of the interface between 2G4 and c-Kit. Residues in the D2 and D3 regions of c-Kit are indicated by shaded bars, respectively. Specific details for implementing the invention

[0264] The present invention will be described in more detail below through embodiments. These embodiments are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments according to the gist of the invention.

[0265] Examples

[0266] Preparation Example

[0267] Preparation Example 1. Preparation of anti-c-Kit antibody 2G4

[0268] Preparation Example 1-1. Preparation of immunized mice

[0269] An emulsion was prepared by mixing 50 μg of recombinant c-Kit protein (cat# PKSH030939) purchased from Elabscience (per mouse) with an equal volume of complete Freund's Adjuvant (Sigma, USA). The emulsion thus prepared was injected intraperitoneally into six 7-week-old female humanized NSG mice produced by human CD34+ cell injection. 50 μg of antigen was injected into each mouse at a total volume of 500 μl. After 1 and 2 weeks, an emulsion containing incomplete Freund's Adjuvant (Sigma, USA) and the antigen was additionally injected intraperitoneally into the mice, respectively.

[0270] Preparation Example 1-2. Confirmation of antibody production

[0271] Blood was collected from the eyes of mice immunized using the above method, placed in a 1.5 ml microcentrifuge tube, and centrifuged at 13,000 rpm for 10 minutes. The serum was separated and stored at -20°C until an experiment to confirm antibody production was performed. After confirming antibody production by performing an enzyme immunoassay using the antigen protein, an emulsion mixed with incomplete Prawn Azuvant (Sigma, USA) and the antigen was injected once more into the peritoneal cavity of the mice three days prior to cell fusion.

[0272] Preparation Examples 1-3. Preparation of Hybridoma

[0273] After confirming antibody production, mice were sacrificed. Splenocytes were isolated and fused with myeloma cells P3X63Ag 8.653 (ATCC CRL-1580) to produce a hybridoma.

[0274] Specifically, mouse P3X63Ag 8.653 cells were cultured in culture plates using RPMI1640 medium supplemented with 10% fetal bovine serum. To perform cell fusion, P3X63Ag 8.653 cells were washed twice with serum-free RPMI1640 medium (Hyclone, USA), and 1 x 10⁶ 7 The concentration was adjusted to the required level. Mice were sacrificed by cervical dislocation, and the spleens were harvested, placed in a mesh container (Sigma, USA), and the cells were isolated. A splenocyte suspension was prepared, and the suspension was washed using centrifugation. Red blood cells were lysed by exposing the splenocyte solution to Tris-NH4Cl (TRIS 20.6 g / L, NH4Cl 8.3 g / L). The completely isolated antibody-producing cells were centrifuged at 400 xg for 5 minutes. Subsequently, they were washed twice in serum-free medium and resuspended in 10 ml of medium. Lymphocytes were counted using a hemocytometer, and 1 x 10⁶ lymphocytes 8 It was mixed with 1 x 10 (10:1) P3X63Ag 8.653 cells in serum-free medium.

[0275] After centrifuging at 400 xg for 5 minutes, 1 ml of 50% (M / V) polyethylene glycol 1500 (Sigma, USA), heated to 37°C, was added dropwise and mixed for 1 minute. The fusion mixture thus prepared was diluted with serum-free RPMI1640 and centrifuged at 400 xg for 3 minutes. Cells were suspended in 35 ml of RPMI1640 selective medium supplemented with 20% fetal bovine serum and HAT (100 μM hypoxanthin, 0.4 μM aminopterin, 16 μM thymidine). 100 μl of the suspension was loaded into a 96-well plate coated with feeder cells (macrophages isolated from the peritoneal cavity using RPMI1640) one day prior and cultured at 37°C and 5% CO2. After 5 days, the HAT medium was replaced every 2 to 3 days, and the cells were cultured for 14 days. After 14 days, the medium was replaced with RPMI1640 medium supplemented with 20% fetal bovine serum and HT (medium from which 0.4 μM aminopterin was removed from HAT) for secondary culture.

[0276] Preparation Example 1-4. Selection and isolation of antibody-producing fusion cells

[0277] The supernatant of the previously prepared fused cell culture medium was collected, and an enzyme immunoassay was performed to confirm whether specific antibodies were produced against the prepared antigen. The culture medium of fused cells exhibiting an appropriate concentration of at least four times that of the negative control group was selected and transferred to a 24-well plate for culture. Additionally, the culture medium was cultured in a 96-well plate at a limiting dilution to accommodate one cell per well, after which the culture medium was recovered and coated with the c-KIT protein used as the antigen at a concentration of 0.1 μg per well in the 96-well plate. Subsequently, an enzyme immunoassay was performed to select fused cells that produced 15 monoclonal antibodies (1C6, 1H2, 1A6, AFA, 2B3, 2G4, 4G5, 4C4, 4C7, 4D7, 1E1, 2H6, 1G3, 1A3, 1D3).

[0278] Preparation Examples 1-5. Selection of c-KIT antibodies

[0279] Surface Plasmon Resonance (SPR) was performed to confirm the c-KIT binding ability of the selected antibodies. Using an SR7500DC (Reichert, USA), 20 μg of human c-Kit (elabscience, PKSH030939), mouse c-Kit (SB, Lot#LC05DE2304), and rat c-Kit (SB, Lot#LC06SE1787), which were used for antibody production, were immobilized on a PEG (Reichert, USA) chip. Subsequently, the antibodies were flowed at different concentrations, and the KD values, which represent the affinity for the c-Kit, were analyzed using the Scrubber2 program. The KD value is calculated by dividing kd by ka; a lower value indicates a higher binding ability to the corresponding target.

[0280] As a result, the KD value of the 2G4 antibody against the human c-Kit was approximately 2.8237 (±0.9) X 10⁻⁶ -12 It showed strong affinity as M. Affinity for humans was the highest, followed by mice and rats.

[0281] The CDR sequences of the selected 2G4 antibodies are as shown in Table 1 below:

[0282] CDR Sequence SEQ ID NO H-CDR1 GFTFSRYG SEQ ID NO: 1 H-CDR2 IWYDGTNK SEQ ID NO: 2 H-CDR3 AREDWAEAFD M SEQ ID NO: 3 L-CDR1 QSLLHSNGYN Y SEQ ID NO: 4 L-CDR2 LGS SEQ ID NO: 5 L-CDR3 MQALQTIT SEQ ID NO: 6

[0283] The sequences of the heavy chain variable region and light chain variable region of the above 2G4 antibody are as shown in Table 2 below:

[0284] Variable region Sequence SEQ ID NO heavy chain QVQLVESGGG VVQPGRSLRL SCAASGFTFS RYGMHWVRQA PGKGLEWVAV IWYDGTNKDY TDSVRGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARED WAEAFDMWGQ GTTVTVSS SEQ ID NO: 7 light chain DIVMTQSPLS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQALQTI TFGQGTRLEI K SEQ ID NO: 8

[0285] Preparation Example 2. Preparation of "2G4-SMCC-DM1 (#POC-DM1)"

[0286] SMCC as an indivisible linker ( N -succinimidyl-4-( N DM1 as a -maleimidomethyl cyclohexane-1-carboxylate) and microtubule inhibitor (N (2')-deacetyl- N A 2G4-ADC (antibody-drug conjugate) containing (2')-(3-mercapto-1-oxopropyl)-maytansine was prepared. The conjugation of the linker-payload to 2G4 was measured using a UV-Vis spectrophotometer, and the drug-to-antibody ratio (DAR) was determined by LC-MS analysis.

[0287] Specifically, 2G4 and human IgG1 (Sino Biological) were dialyzed against conjugation buffer (0.1 M sodium phosphate and 0.15 M NaCl, pH 7.2) and conjugated with SMCC-DM1 (MedChemExpress) at a molar ratio of 1:5 or 1:10 for 1.5 hours at RT. Fractionation was performed to remove unbound SMCC-DM1 and aggregated 2G4-DM1. The antibody fractions were pooled and dialyzed against formulation buffer (10 mM sodium succinate, 0.05% polysorbate 20, and 6% sucrose; pH 5.0). Conjugation was determined by examining optical density (OD) at 280 nm and 252 nm using a SPECTROstar Nano (BMG LABTECH). DAR was determined using liquid chromatography-tandem mass spectrometry (LC-MS).

[0288] To confirm the conjugation of DM1 and 2G4, since DM1 absorbs ultraviolet light at 252 nm, the absorbance of Naked 2G4 and ADC at 252 nm was analyzed using a UV-Vis spectrophotometer, and the absorbance of Naked 2G4 and 2G4-DM1 was compared. The absorbance of 2G4-DM1 was higher than that of 2G4 at 252 nm. The results indicated that DM1 was potentially conjugated to 2G4.

[0289] Mass spectrometry of 2G4-DM1 confirmed that it consists of various populations with drug ranges from 1 to 4 per antibody, and the average DAR of 2G4-DM1 is 1.8.

[0290] Preparation Example 3. Preparation of an ADC using a cuttable linker

[0291] Four types of ADCs were fabricated using a cleavable linker. Specifically, the ADCs utilized classical maleimide conjugation or ThioBridge to combine MMAE and SN-38. ® It was manufactured using disulfide rebridging conjugation technology (Abzena, USA), and the average DAR range was between 2 and 8. ThioBridge with an average DAR of 2 and 4 in ratio. ® MMAE ADCs were prepared together with maleimide MMAE control ADCs having an average DAR of 4. ThioBridge ® The mean DAR of the SN-38 ADC was 8. A cathepsin-cleavable 'Val-Cit-PAB' motif was included in all linkers to allow lysosome release of the payload (Fig. 1a). ThioBridge ® In the case of SN-38 ADCs, the carbonate bond connecting SN-38 to the linker is sensitive to hydrolysis, enabling a pH-based payload release mechanism. Considering subsequent biological testing, ADCs with high monomer purity (>97%) were prepared in amounts of 29–56 mg. ThioBridge ® The manufacturing process of -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) is as shown in Fig. 1b.

[0292] Preparation Example 3-1. Preparation of 2G4-MC-VC-PAB-MMAE

[0293] 13.38 mg / mL of mAb 2G4 in Dulbecco's PBS, pH 7.4, 5 mM EDTA (5.232 mL; 70.0 mg; 470 nmol; 1.0 equivalent) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (8.505 mL). A 5 mM solution of TCEP dissolved in endotoxin-free water (263.4 μL, 1317 nmol, 2.8 equivalents) was added to the mAb 2G4 dilution. Reduction was carried out at 40°C for 2 hours at a final antibody concentration of 5.0 mg / mL.

[0294] After 2 hours at 40°C, the reduction mixture was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (2.450 mL), cooled to 22°C, and then further diluted with DMF (177 μL). The 4.26 mg / mL (3.23 mM) solution of MC-VC-PAB-MMAE in DMF corresponded to 3.95 mg (3.00 μmol) of MC-VC-PAB-MMAE (MW = 1317 g·mol -1 ) was prepared by dissolving in 927 μL of DMF. The MC-VC-PAB-MMAE solution (873 μL; 3.72 mg; 2823 nmol; 6.0 equivalents) in DMF was added to the reduced mAb 2G4 solution to obtain a final concentration of 6% DMF and a final antibody concentration of 4.0 mg / mL. The conjugation reaction was carried out at 22°C for 1 hour.

[0295] After 1 hour at 22°C, buffer exchange was performed on the reaction mixture by spin filtration using a Zebaspin column (7 kDa MWCO; 10 mL) equilibrated to 20 mM histidine, 50 mM NaCl, 5% sucrose, and pH 6.5 according to the manufacturer's instructions. The recovered conjugate sample was further polished and concentrated to 7.13 mg / mL by ultrafiltration / diafiltration using a Vivaspin 20 centrifuge concentrator (PES membrane, 30 kDa MWCO) equilibrated to 20 mM histidine, 50 mM NaCl, 5% sucrose, and pH 6.5. The concentrated conjugate sample (56.3 mg; 7.90 mL) was sterile filtered through a PVDF membrane filter with a pore size of 0.22 μm.

[0296] The antibody-drug conjugate obtained through this process was named "2G4-MC-VC-PAB-MMAE" and characterized by HIC and SEC. 56 mg of 2G4-MC-VC-PAB-MMAE ADC was isolated with a high monomer content (>98%, SEC) and an average DAR of 4.2 (HIC) (hereinafter referred to as 003-2).

[0297] Preparation Example 3-2. 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2 manufacturing

[0298] 14.77 mg / mL of mAb 2G4 in Dulbecco's PBS, pH 7.4, 5 mM EDTA (2.180 mL; 32.2 mg; 216 nmol; 1.0 equivalent) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (4.148 mL). A 5 mM solution of TCEP dissolved in endotoxin-free water (112 μL, 562 nmol, 2.6 equivalents) was added to the diluted mAb 2G4 solution. Reduction was carried out at 40°C for 2 hours at a final antibody concentration of 5.0 mg / mL.

[0299] After 2 hours at 40°C, the reduction mixture was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (805 μL), cooled to 22°C, and then further diluted with DMF (403 μL). ThioBridge in DMF ® A 4.52 mg / mL (1.61 mM) solution of -Glu-(Val-Cit-PAB-MMAE)-PEG (24 u) was used with 2.15 mg (768 nmol) of ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u)(MW = 2805.0 g.mol -1 ThioBridge in DMF was prepared by dissolving ) in 477 μL of DMF. ® -Glu-(Val-Cit-PAB-MMAE)-PEG (24u) solution (402 μL; 1.82 mg; 648 nmol; 3.0 eq.) was added to the reduced 2G4 solution to obtain a final concentration of 10% DMF and a final antibody concentration of 4.0 mg / mL. The conjugation reaction was allowed to proceed at 22°C for 18 hours.

[0300] After 18 hours at 22°C, buffer exchange was performed on the reaction mixture by spin filtration using a Zebaspin column (7 kDa MWCO; 10 mL) equilibrated to 20 mM histidine, 50 mM NaCl, 5% sucrose, and pH 6.5 according to the manufacturer's instructions. The recovered conjugate sample was further polished and concentrated to 7.25 mg / mL by ultrafiltration / diafiltration using a Vivaspin 20 centrifuge (PES membrane, 30 kDa MWCO) equilibrated to 20 mM histidine, 50 mM NaCl, 5% sucrose, and pH 6.5. The concentrated conjugate sample (29.7 mg; 4.10 mL) was sterile filtered through a PVDF membrane filter with a pore size of 0.22 μm.

[0301] The antibody-drug conjugate obtained through this process is "2G4-ThioBridge" ®It was named -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2", and the conjugate was characterized by HIC and SEC. 30 mg of 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR2 ADC was isolated with a high monomer content (>97%, SEC) and an average DAR of 2.0 (HIC). (Hereinafter referred to as 003-1)

[0302] Preparation Example 3-3. 2G4-ThioBridge ® Manufacture of Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4

[0303] 20.71 mg / mL of mAb 2G4 in Dulbecco's PBS, pH 7.4, 5 mM EDTA (5.794 mL, 120 mg, 806 nmol, 1.0 equivalent) was diluted with 17 mL of Dulbecco's PBS, pH 7.4, 5 mM EDTA. A 5 mM solution of TCEP dissolved in endotoxin-free water (967.2 μL, 4836 nmol, 6.0 equivalent) was added to the diluted mAb 2G4 solution. Reduction was carried out at 40°C for 1 hour at a final antibody concentration of 5.0 mg / mL.

[0304] After 1 hour at 40°C, the reduction mixture was diluted with propylene glycol (6.40 mL) and cooled to 22°C. ThioBridge in DMF ® An 8.48 mg / mL (3.02 mM) solution of -Glu-(Val-Cit-PAB-MMAE)-PEG (24u) was ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (MW = 2805.0 g.mol -1 It was prepared by dissolving 20.60 mg (7345 nmol) of ) in 2430 μL of DMF. ThioBridge in DMF ®-Glu-(Val-Cit-PAB-MMAE)-PEG (24 μL) solution (1600 μL; 13.57 mg; 4836 nmol; 6.0 equivalents) was added to the reduced 2G4 solution to produce 5% DMF, 20% propylene glycol, and a final antibody concentration of 3.8 mg / mL. The conjugation reaction was allowed to proceed at 22°C for 18 hours.

[0305] After 18 hours at 22°C, the reaction mixture was diluted with 4 M sodium chloride, 50 mM sodium phosphate, pH 7.0 (32.0 mL). The diluted reaction mixture was loaded onto a 10 mL Proteus FliQ column packed with HIC ToyoPearl® Phenyl-650S resin, equilibrated with Buffer A (2.0 M sodium chloride, 50 mM sodium phosphate, pH 7.0 buffer). Elution was performed in Buffer B (50 mM sodium phosphate, 20% isopropanol, pH 7.0) at a constant flow of 1.5 mL / min and a 200 mL gradient of 0–100%. Fractions were analyzed by HIC and SEC and pooled based on the content of four DAR species (>90%). The pooled fraction was buffered and concentrated to 7.93 mg / mL by ultrafiltration / diafiltration using a Vivaspin 20 centrifuge (PES membrane, 30 kDa MWCO) equilibrated to 20 mM histidine, 50 mM NaCl, 5% sucrose, and pH 6.5. The concentrated conjugate sample (47.4 mg; 5.98 mL) was sterile filtered through a PVDF membrane filter with a pore size of 0.22 μm.

[0306] The antibody-drug conjugate obtained through this process is "2G4-ThioBridge" ® It was named "-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4 conjugate", and said conjugate was characterized by HIC and SEC. 47 mg of 2G4-ThioBridge ®-Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR4 ADC was isolated with a high monomer content (>98%, SEC) and an average DAR of 4.0 (HIC) (hereinafter referred to as 004-2).

[0307] Preparation Examples 3-4. 2G4-ThioBridge ® Preparation of -Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2

[0308] 18.71 mg / mL of mAb 2G4 in Dulbecco's PBS, pH 7.4, 5 mM EDTA (3.474 mL, 65.0 mg, 437 nmol, 1.0 equivalent) was diluted with Dulbecco's PBS, pH 7.4, 5 mM EDTA (9.00 mL). A 5 mM solution of TCEP dissolved in endotoxin-free water (524.0 μL; 2620 nmol, 6.0 equivalent) was added to the diluted mAb 2G4 solution. Reduction was carried out at 40°C for 1 hour at a final antibody concentration of 5.0 mg / mL.

[0309] After 1 hour at 40°C, the reduction mixture was diluted with propylene glycol (3.71 mL), cooled to 22°C, and then further diluted with DMF (655 μL). ThioBridge in DMF ® A 10.0 mg / mL (2.18 mM) solution of -Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 is 14.09 mg (3072 nmol) of ThioBridge ® -Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 (MW = 4588.0 g.mol -1 ThioBridge was prepared by dissolving ) in 1409 μL of DMF. In the DMF solution (1202 μL; 12.02 mg; 2620 nmol; 6.0 equivalents), ThioBridge ®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 was added to the reduced 2G4 solution to produce a final concentration of 10% DMF, 20% propylene glycol, and a final antibody concentration of 3.5 mg / mL. The conjugation reaction was allowed to proceed at 22°C for 18 hours.

[0310] After 18 hours at 22°C, the reaction mixture was diluted with 4 M sodium chloride, 50 mM sodium phosphate, pH 7.0 (18.6 mL). The diluted reaction mixture was equilibrated with HIC ToyoPearl in Buffer A (2.0 M sodium chloride, 50 mM sodium phosphate, pH 7.0 buffer). ® It was loaded onto a 10 mL Proteus FliQ column packed with Phenyl-650S resin. Elution was performed in Buffer B (50 mM sodium phosphate, 20% isopropanol, pH 7.0) at a constant flow of 1.5 mL / min and a 200 mL gradient of 0–100%. Fractions were analyzed by HIC and SEC and pooled according to the content of four DAR species (>80%). The pooled fractions were concentrated to 4.01 mg / mL by buffer exchange via ultrafiltration / diafiltration using a Vivaspin 20 centrifuge (PES membrane, 30 kDa MWCO) equilibrated to 20 mM histidine, 50 mM NaCl, 5% sucrose, and pH 6.5. A concentrated conjugate sample (29.0 mg; 7.22 mL) was sterile filtered through a PVDF membrane filter with a pore size of 0.22 μm.

[0311] The antibody-drug conjugate obtained through this process is "2G4-ThioBridge" ® It was named "-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 conjugate", and the said conjugate was characterized by LC-MS and SEC. 29 mg of 2G4-ThioBridge ®-Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 was separated with a high monomer content (>98%, SEC) and an average DAR of 7.4 (LC-MS) (hereinafter referred to as 007-1).

[0312] Analysis method

[0313] (1) LC-MS analysis

[0314] LC-MS analysis was performed using a POROSHELL 300SB C3 column (2.1 x 12.5 mm, 5 μm) connected to a Waters XEVO G2S TOF mass spectrometer and a Waters Acquity H Class UPLC system. The mobile phase was Buffer A (0.1% formic acid in water). A gradient (10% B for 2.5 min, 10-80% B gradient for 3.5 min) was applied using Buffer B (acetonitrile, 0.1% formic acid) at a flow rate of 0.4 mL / min. The column was maintained at 60°C throughout the analysis. Maleimide ADCs were analyzed after reduction (10 mM DTT, at 40°C for 1 hour). All ADCs were analyzed after dilution to 0.2 mg / mL. 10 μL of ADC solution was injected for analysis. The average DAR was calculated as the weighted average of the observed DAR species based on the major glycoform signal intensity (SI) of the deconvolved m / z spectrum for non-reducing samples and the signal intensity for light chains (LSI) and heavy chains (HSI) for reducing samples:

[0315]

[0316] (2) SEC Analysis

[0317] The analytical SEC was performed using an ACQUITY UPLC BEH SEC column (4.6 mm x 15 cm, 200 Å, 1.7 μm) and a protection column (4.6 mm x 3 cm) connected to a Dionex Ultimate 3000 UPLC system. The mobile phase consisted of 0.2 M potassium phosphate buffer, pH 6.8, 0.2 M potassium chloride, and 15% (v / v) isopropanol. The flow rate was maintained constant at 0.35 mL / min. The column was maintained at 30°C throughout the analysis. The analysis was performed with 10-minute isosolvent elution using UV detection at 248 nm, 280 nm, and 365 nm. 10 μg of ADC was injected for the analysis. The percentage of high molecular weight (HMW) species was calculated by comparing the peak area corresponding to HWM species at 280 nm with the total peak area corresponding to both HWM and monomeric species at 280 nm. The presence of free reagent-related species was evaluated by comparing the chromatographic traces of the ADC sample and buffer sample in the low molecular weight species region of the chromatogram for a retention time of more than 6 minutes at the wavelength corresponding to the maximum absorption of the payload (λ = 248 nm for MMAE, λ = 365 nm for SN-38).

[0318] (3) HIC analysis

[0319] The HIC analysis was performed using a TOSOH Bioscience TSKgel Butyl-NPR column (4.6 mm x 3.5 cm, 2.5 μm) connected to a Dionex Ultimate 3000 UPLC system. The mobile phase was Buffer A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. To elute the bound species, a linear gradient (0–100% B within 10.5 min) was applied using Buffer B (20% isopropanol, 50 mM sodium phosphate, pH 7.0) at a flow rate of 1.35 mL / min. The column was maintained at °C throughout the analysis. The analysis was performed using UV detection at 280 nm. 10 μg of ADC was injected per analysis. The percentage (i) of each DAR species was calculated by comparing the peak area of ​​each assigned peak to the total peak area. The average DAR was calculated as the weighted average of the observed DAR species based on the peak area under the curve (AUCi), and the average molecular weight of the ADC was calculated as follows based on the DAR and linker-payload mass contributions:

[0320]

[0321] Examples

[0322] Example 1. In a test tube ( in vitro Cell viability analysis

[0323] In vitro cell viability analysis was performed to confirm the efficacy of the ADCs shown in Table 3 in c-Kit-positive and c-Kit-negative cancer cell lines.

[0324] Cells were seeded into 96-well cell culture black plates (Greiner Bio-One, Kremsmuenster, Austria), and cell counts were scanned using a Celigo Imaging Cytometer. Nine concentration points of the ADC were prepared by serially diluting 5- or 10-fold starting concentrations of 40 or 200 μg / ml. After 3–5 days of treatment, cells were stained with Calcein-AM (1 μg / ml) or Hoechst 33342 (8 μM) fluorescent dyes. Wells were scanned and analyzed using a Celigo Imaging Cytometer to count viable cells. Cell viability was graphed using Graph Pad Prism Software, and IC50 values ​​were calculated (Table 4).

[0325] Name Seeding number Max Conc. Dilution fold Incubation time GIST-T1 5Х10 3 / well 40 μg / mL 10 3 days GIST-430 / 654 5Х10 3 / well 40 μg / mL 10 4 days MDA-MB-468 4Х10 3 / well 200 μg / mL 5 3 days NCI-H526 7Х10 3 / well 200 μg / mL 5 5 days HMC1.2 5Х10 3 / well 200 μg / mL 5 4 days Kasumi-1 2Х10 4 / well 200 μg / mL 5 4 days

[0326] ADC Batch Code IC50 (μg / ml) GIST-T1 GIST-430 / 654 HMC1.2 MDA-MB-468 2G4-SMCC-DM1 POC-DM1 0.00451 0.0033 0.1636 1.246 2G4-MC-Val-Cit-PAB-MMAE NOVN-1574-JN003-2 0.00367 0.0058 0.005 6.256 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2 NOVN-1614-JN003-1 0.00745 0.0097 0.0097 44.92 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4 NOVN-1574-JN004-2 0.00314 0.0051 0.0054 25.84 2G4-ThioBridge ® -Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2 NOVN-1574-JN007-1 0.0589 0.0546 0.095 0.168

[0327] As shown in Figure 2, it can be seen that the ADC group shows statistically significant efficacy compared to the control group.

[0328] Example 2. In vivo (GIST T1 cells) against GIST in mice (GIST T1 cells) in vivo ) Benefits

[0329] Experiments were conducted to evaluate the efficacy of ADC substances against GIST. A GIST-T1 cell (c-Kit mutant and imatinib-sensitive; accession number: CVCL_4976; c-Kit mutation site - Exon11 V560-L576) xenograft model was used.

[0330] Four-week-old female CB-17 SCID immunodeficient mice (Charles River Laboratories Japan, Inc.) were acclimatized to Specific Pathogen Free (SPF) conditions for 12 days before being used in the experiment. GIST-T1 cells (5 x 10⁶) in 50% Matrigel (Corning, 354248, NY, USA) subcutaneously were placed in six-week-old mice. 6 100 μL of Head / Serum-free DMEM / High glucose medium was inoculated. The total injection volume containing suspended cells was 200 μL.

[0331] The average tumor volume on day 21 after transplantation was approximately 190 mm² 3 Mice were registered on Day 0. After being randomly assigned to one of six groups (n = 5 / group), mice were administered an ADC vehicle (5 ml / kg, iv tail), each ADC substance (0.5, 1.5, or 3.0 mg / kg, iv tail), imatinib (100 mg / kg, po), and an ADC substance (3.0 mg / kg, iv tail) + imatinib (100 mg / kg, po) (Table 5). The buffer for PoC-DM1 as the ADC vehicle was a mixture of 10 mM sodium succinate, 6% sucrose, and 0.05% Tween20 (pH 5.0), and the buffer for the remainder was a mixture of 20 mM histidine (Merck, 104352), 50 mM sodium chloride (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5), and 0.22 μM filter (Merck S2GPU11RE). Individual doses were calculated based on the animal body weight recorded immediately before administration, with a dose volume of 5 mL / kg body weight.

[0332] No. Subject Interval, Route 1 Control (vehicle, ADC buffer) D0, D10, D20 (Three times), iv 2 Imatinib 100 mg / kg D0 - D29 (Daily for 30 days), po 3 ADC 0.5 mg / kg D0, D10, D20 (Three times), iv 4 ADC 1.5 mg / kg D0, D10, D20 (Three times), iv 5 ADC 3.0 mg / kg D0, D10, D20 (Three times), iv 6 ADC 3.0 mg / kg + Imatinib 100 mg / kg ADC: D0, D10, D20 (Three times), ivImatinib: D0 - D29 (Daily for 30 days), po

[0333] The administered ADC substances are as shown in Table 6:

[0334] Study # ADC #PoC-DM1 2G4-SMCC-DM1 #003-1 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2 #004-2 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4 #003-2 2G4-MC-Val-Cit-PAB-MMAE

[0335] All animals were observed once daily for mortality, general condition, and clinical signs (time, onset, severity, and recovery). Tumor volume and body weight were measured twice a week. To measure tumor volume, calipers were used to measure the length, width, and depth.

[0336] The results are shown in Figures 3a to 3d.

[0337] No adverse events were observed in any of the groups except the imatinib 100 mg / kg group during the study period prior to sacrifice. In the imatinib 100 mg / kg group, one case of death occurred on Day 91.

[0338] Compared to the control group (vehicle), no weight loss or weight gain of more than 10% was observed in the imatinib 100 mg / kg group and the test ADC study groups (0.5 mg / kg, 1.5 mg / kg, 3.0 mg / kg, 3.0 mg / kg + imatinib 100 mg / kg) for 56 days. Subsequently, no rapid weight loss or rapid weight gain was observed in any group. This implies that all of the inventors' ADC materials are very safe.

[0339] On day 56, compared to the control group (vehicle), the imatinib 100 mg / kg group inhibited tumor growth, whereas all 3.0 mg / kg ADC study groups showed a stronger tumor growth inhibitory effect. In particular, 2G4 ThioBridge ® The ADC study group of -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) showed a significant tumor growth inhibitory effect even at 1.5 mg / kg.

[0340] Although imatinib alone suppressed the tumor, the tumor recurred after treatment was discontinued. Surprisingly, combination therapy with each ADC 3 mg / kg and imatinib induced complete remission of the tumor without regrowth for up to 105 or 112 days after discontinuation of imatinib administration.

[0341] As a result of measuring tumor weight after sacrifice, the imatinib 100 mg / kg group showed a reduction of approximately 59% compared to the control group (vehicle), whereas all ADC study groups administered 1.5 mg / kg or higher (excluding the #PoC-DM1 1.5 mg / kg group) demonstrated a higher reduction ability (Table 7). In particular, 2G4 ThioBridge ® The ADC study groups of -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) demonstrated significant tumor growth inhibitory efficacy even in the 1.5 mg / kg group. When each ADC was administered at 3 mg / kg in combination with imatinib, most tumors disappeared.

[0342] (reduction %) #PoC-DM1 #003-1 #004-2 #003-2 0.5 mg / kg 0.7% 32.0% 40.6% 45.4% 1.5 mg / kg 20.3% 85.0% 98.1% 61.5% 3.0 mg / kg 70.0% 89.8% 99.2% 93.8% 3.0 mg / kg + imatinib 100 mg / kg 98.6% 98.8% 99.2% 94.7%

[0343] The above results support the fact that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0344] Example 3: In vivo efficacy of GIST in mice (GIST-430 / 654 cells)

[0345] In particular, experiments were also conducted to evaluate the efficacy of ADC substances against GIST resistant to imatinib. A GIST-430 / 654 cell (imatinib resistant, Exon 11 V560-L576 + Exon 13 V654A) xenograft model was used.

[0346] 5-week-old female NOG (NOD / Shi-scid, IL-2RγKO) immunodeficient mice (CIEA Japan, Inc.) were acclimatized for 12 days under Specific Pathogen Free (SPF) conditions before use in the experiment. GIST-430 / 654 cells (5×10⁻¹⁰) in 50% Matrigel (Corning, 354248, NY, USA) were subcutaneously subcutaneously in 6-week-old mice. 6 100 μL of Head / Serum-free IMDM medium was injected subcutaneously. The total injection volume containing suspended cells was 200 μL.

[0347] Mice were enrolled in this study on day 21 after transplantation, and the average tumor volume was approximately 180 mm³. 3 (Day 0). After randomizing into one of five groups (n = 5 / group), mice were administered an ADC vehicle (5 ml / kg, iv tail), each ADC substance (1, 3, or 5 mg / kg, iv tail), and imatinib (100 mg / kg, po) (Table 8). The buffer for PoC-DM1 as the ADC vehicle was a mixture of 10 mM sodium succinate, 6% sucrose, and 0.05% Tween20 (pH 5.0), and the buffer for the remainder was a mixture of 20 mM histidine (Merck, 104352), 50 mM NaCl (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5), and 0.22 μM filter (Merck, S2GPU11RE). The individual dose was calculated based on the animal's body weight recorded immediately before administration, with a dose volume of 5 mL / kg body weight.

[0348] No. Subject Interval, Route 1 Control (vehicle, ADC buffer) D0, D7, D14 (Three times), iv 2 Imatinib 100 mg / kg D0 - D29 (Daily for 30 days), po 3 ADC 1 mg / kg D0, D7, D14 (Three times), iv 4 ADC 3 mg / kg D0, D7, D14 (Three times), iv 5 ADC 5 mg / kg D0, D7, D14 (Three times), iv

[0349] The ADC materials are as shown in Table 9 below:

[0350] Study # ADC #PoC-DM1 2G4-SMCC-DM1 #003-1 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 2 #004-2 2G4-ThioBridge ® -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4 #003-2 2G4-MC-Val-Cit-PAB-MMAE #007-1 2G4-ThioBridge ® -Glu-[(Val-Cit-PAB-SN-38)]2-Glu-[PEG(24u)]2

[0351] The sacrifice dates for each group were as follows:

[0352] - Vehicle and imatinib 100 mg / kg: Sacrificed on day 49.

[0353] - All ADC groups (except 007-1): Sacrificed on day 63.

[0354] - Group #007-1: Sacrificed on day 42 (No comparison was made as Group #007-1 ended earlier than the control group (vehicle)).

[0355] All animals were observed once daily for mortality, general condition, and clinical signs (time, onset, severity, and recovery). Tumor volume and body weight were measured twice a week. To measure tumor volume, calipers were used to measure length, width, and depth.

[0356] The results are shown in Figures 4a to 4e.

[0357] No abnormal symptoms were observed in any group during the test period prior to sacrifice.

[0358] Between the imatinib 100 mg / kg group and the test ADC study groups (1 mg / kg, 3 mg / kg, and 5 mg / kg), no weight loss or weight gain of more than 10% was observed over 49 days compared to the control group (vehicle). Subsequently, no rapid weight loss or rapid weight gain was observed in any group. This implies that all ADC substances of the present invention are very safe.

[0359] At day 49, compared to the control group (vehicle), the imatinib 100 mg / kg group showed almost no inhibition of tumor growth, whereas all ADC study groups demonstrated a stronger tumor growth inhibitory effect (except for #007-1 1 mg / kg). In particular, 2G4 ThioBridge ®The ADC study group of -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) was found to have a significant inhibitory effect on tumor growth in the 3 or 5 mg / kg groups, and the tumors remained at nearly the same level even after treatment was discontinued.

[0360] Tumor weight measurements after sacrifice showed that all study groups (except #007-1; terminated earlier than the control group) exhibited a greater reduction compared to the control group (vehicle) (see Table 10 below). In particular, 2G4 ThioBridge ® The ADC study group of -Glu-(Val-Cit-PAB-MMAE)-PEG(24u) (#003-1 and #004-2) was found to have a significant tumor growth inhibitory effect.

[0361] (reduction %) #PoC-DM1 #003-1 #004-2 #003-2 3 mg / kg 23.9 49.1 78.0 34.2 5 mg / kg 17.6 67.3 87.4 70.2

[0362] The above results support the fact that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0363] Example 4: In vivo efficacy of SCLC in mice (SCLC-H526 cells)

[0364] Experiments were conducted to evaluate the efficacy of ADC substances against SCLC. An SCLC-H526 (NCI-H526, stage E, carcinoma; variant small cell lung cancer, c-Kit overexpression) xenograft model was used.

[0365] The above analysis was performed on SCLC-H526 cells (2 x 10⁶) in 50% Matrigel (Corning, 354248, NY, USA). 6 The procedure was performed in the same manner as in Example 2, except that 100 μL of Head / Serum-free RPMI-1640 medium was used. Mice were enrolled in the study on day 13 after transplantation, and the average tumor volume was approximately 165 mm². 3(Day 0); imatinib was administered orally for 22 days from D0 to D21. The study groups were #PoC-DM1, #003-1, #004-2 and #003-2.

[0366] The above results are shown in FIGS. 5a to 5d.

[0367] No abnormal symptoms were observed in the entire group during the test period prior to the sacrifice.

[0368] Between the imatinib 100 mg / kg group and the test ADC study groups (1 mg / kg, 3 mg / kg, and 5 mg / kg), no weight loss or weight gain of more than 10% was observed over 21 days compared to the control group (vehicle). Subsequently, no rapid weight loss or rapid weight gain was observed in any group. This implies that all ADC substances of the present invention are very safe.

[0369] On day 21, compared to the control group (vehicle), the imatinib 100 mg / kg group showed almost no inhibition of tumor growth, whereas the 3 or 5 mg / kg ADC study groups showed a stronger tumor growth inhibitory effect (excluding #007-1 1 mg / kg). Notably, treatment with the #004-2 3 mg / kg and #004-2 5 mg / kg groups induced complete remission of the tumor without regrowth even after discontinuation of administration for up to 105 days.

[0370] As a result of measuring tumor weight after sacrifice, all study groups (except #007-1; terminated earlier than the control group) showed a greater reduction compared to the control group (vehicle) (see Table 11 below). In particular, 2G4 ThioBridge ® The ADC study group of -Glu-(Val-Cit-PAB-MMAE)-PEG(24u)(#004-2) showed a significant tumor growth inhibitory effect.

[0371] (reduction %) #PoC-DM1 #003-1 #004-2 #003-2 5 mg / kg 73.8 60.0 99.5 53.7

[0372] The above results support the fact that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0373] Example 5: In vivo efficacy against mast cell tumors in mice (HMC 1.2 cells)

[0374] NN3201 (2G4-ThioBridge) for mast cell tumor ® Experiments were performed to evaluate the efficacy of -Glu(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4; #004-2). An HMC 1.2 (c-Kit mutation and imatinib resistance; Exon11(V560G) + Exon17(D816V)) xenograft model was used.

[0375] Four-week-old female CB-17 SCID immunodeficient mice (Janvier France, Inc.) were acclimatized for 18 days under Specific Pathogen Free (SPF) conditions and used in the experiment. HMC-1.2 cells in 50% Matrigel (Corning, 354248, NY, USA) were subcutaneously injected into six-week-old mice (1 x 10⁻¹⁰). 6 / 100 μL / Head / Serum-free, IMDM medium). The total injection volume containing suspended cells was 200 μL.

[0376] Mice were enrolled in the study on the 11th day after transplantation, and the average tumor volume was approximately 175.4 mm². 3(Day 0). After randomization to each group (n = 6 / group), mice were administered an ADC vehicle (5 ml / kg, iv tail), 2G4 (5 mg / kg, iv tail), #PoC-DM1 (5 mg / kg, iv tail), NN3201 (1, 3, or 5 mg / kg, iv tail), and imatinib (100 mg / kg, po). The ADC vehicle consisted of a mixture of 20 mM histidine (Merck, 104352), 50 mM NaCl (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5), and 0.22 μM filter (Merck, S2GPU11RE). Individual doses were calculated based on the animal body weight recorded immediately before administration, with a dose volume of 5 mL / kg body weight. IV injections were administered three times on D0, D7, and D14, and imatinib was administered orally for 18 days from D0 to D17.

[0377] The results are shown in Figure 6.

[0378] No abnormal symptoms were observed in any group during the test period.

[0379] Over 17 days, no weight loss or weight gain of more than 10% was observed between the imatinib 100 mg / kg group and the test group compared to the control group (vehicle). Subsequently, no rapid weight loss or rapid weight gain was observed in either group. This implies that all ADC substances of the present invention are very safe.

[0380] On day 17, the imatinib 100 mg / kg group barely inhibited tumors, whereas the ADC study group showed a more potent tumor proliferation inhibitory effect compared to the control group (vehicle).

[0381] Tumors were re-inoculated on day 77. On day 95, compared to the control group (first-) inoculation group, TGI (tumor growth inhibition) was 71.0% in the #NN3201 1 mg / kg (re-) inoculation group, 79.0% in the #NN3201 3 mg / kg (re-) inoculation group, and 74.8% in the #NN3201 5 mg / kg (re-) inoculation group. Additionally, the #PoC-DM1 5 mg / kg (re-) inoculation group showed 46.2% tumor inhibition (Fig. 7).

[0382] The above results support the fact that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0383] Example 6: In vivo efficacy against AML in mice (Kasumi-1 cells)

[0384] This experiment uses the NN3201 (2G4 ThioBridge) for AML ® It was performed to evaluate the efficacy of -Glu(Val-Cit-PAB-MMAE)-PEG(24u) DAR 4). A Kasumi-1 (c-Kit mutant and partially imatinib sensitive; Exon17(N822K)) xenograft model was used.

[0385] The above experiment was conducted on Kasumi-1 cells (3 x 10⁻⁶) in 50% Matrigel (Corning, 354248, NY, USA). 6 The procedure was performed in the same manner as in Example 5, except that 100 μL of Head / Serum-free RPMI1640 medium was used. Mice were enrolled in the study on day 29 after transplantation, and the average tumor volume was approximately 170 mm³. 3 It was (Day 0).

[0386] The results are shown in Figure 8.

[0387] No abnormal symptoms were observed in any group during the test period.

[0388] Over 17 days, no weight loss or weight gain of more than 10% was observed between the imatinib 100 mg / kg group and the test group compared to the control group (vehicle). Subsequently, no rapid weight loss or rapid weight gain was observed in either group. This implies that all ADC substances of the present invention are very safe.

[0389] On day 17, compared to the control group (vehicle), the imatinib 100 mg / kg group barely inhibited tumor growth, and the PoC-DM1 group showed a weak inhibitory effect. The NN3201 group was found to have a stronger tumor growth inhibitory effect.

[0390] The above results support the fact that the ADC group demonstrated statistically significant efficacy compared to the control group.

[0391] Example 7: In vivo efficacy against breast cancer in mice (MDA-MB-468 cells)

[0392] Experiments were conducted to evaluate the efficacy of ADC substances against breast cancer. An MDA-MB-468 (c-Kit, Negative) xenograft model was used in this experiment.

[0393] The above experiment involved MDA-MB-468 cells (5 x 10⁻⁶) in 50% Matrigel (Corning, 354248, NY, USA). 6 The procedure was performed in the same manner as in Example 5, except that 100 μL of Head / Serum-free DMEM medium was used. Mice were enrolled in the study on day 21 after transplantation, and the average tumor volume was approximately 167 mm². 3 It was (Day 0).

[0394] The results are shown in Figures 9a to 9d.

[0395] No abnormal symptoms were observed in any group during the test period prior to sacrifice.

[0396] Compared to the control group (vehicle), no weight loss or weight gain of more than 10% was observed between the imatinib 100 mg / kg group and the test ADC study groups (1 mg / kg, 3 mg / kg, and 5 mg / kg) over 49 days. Subsequently, no rapid weight loss or rapid weight gain was observed in any group. This implies that all ADC substances of the present invention are very safe.

[0397] On day 49, compared to the control group (vehicle), #PoC-DM1 showed no antitumor activity in xenografts transplanted with MDA-MB-468 cells lacking c-Kit expression; on the other hand, #003-1, #004-2, and #003-2 were found to have stronger tumor growth inhibitory effects at all doses. In particular, the group treated with 5 mg / kg of #004-2 was found to significantly inhibit tumor growth.

[0398] As a result of measuring tumor weight after sacrifice, tumor reduction was observed in #003-1, #004-2, and #003-2 compared to the control group (vehicle) (see Table 12 below). In particular, group #004-2 was found to have a significant tumor growth inhibitory effect.

[0399] (reduction %) #003-1 #004-2 #003-2 5 mg / kg 41.2 84.6 62.0

[0400] An interesting finding from the MDA-MB-468 results is that ADCs utilizing 2G4, a cleavable linker, and MMAE exhibited dose-dependent efficacy in c-Kit negative cell-based xenograft models, whereas ADCs utilizing a non-cleavable linker did not. These results suggest that ADCs containing 2G4, a cleavable linker, and MMAE can be ideal partners even when targeting non-c-Kit cells.

[0401] Example 8: In vivo efficacy of SCLC in mice (SCLC-H526 cells) - Re-challenge analysis

[0402] A tumor re-challenge analysis was performed to determine whether NN3201 treatment is effective even after re-inoculation of the tumor.

[0403] The analysis was performed in the same manner as in Example 3, except that the experimental group consisted solely of #004-2 at 3 mg / kg and 5 mg / kg. Tumor re-challenge was performed at the time of complete tumor remission (day 63). The control cohort (square) consisted of animals of the same age used as controls that received the first tumor inoculation on day 63 without initial tumor grafting on the right side. For xenograft analysis, SCLC-H526 (2 x 10⁻¹⁰) in 50% Matrigel (Corning, 354248, NY, USA) was used. 6 Cells (100 μL / Head / Serum-free, RPMI-1640 medium) were injected into CB-17 SCID mice via sc (subcutaneous, left side).

[0404] The results are shown in Figure 10.

[0405] In the tumor re-challenge, the #004-2 groups at 3 mg / kg and 5 mg / kg showed dose-dependent efficacy without additional ADC injections, whereas the control group resulted in tumor growth.

[0406] Example 9: Obesity in mice (HMC 1.2 cells) cell tumor In vivo efficacy analysis of

[0407] A tumor re-challenge analysis was performed to determine whether NN3201 treatment is effective even after re-inoculation of the tumor.

[0408] Tumor re-challenge analysis was performed in the NN3201 (1, 3, or 5 mg / kg, iv tail) group at the time when complete tumor remission was confirmed in Example 4 (Day 77). The inventors included a PoC DM1 ADC to understand the difference between DM1 and MMAE as payloads in terms of immunogenic cell death (ICD). Animals of the same age were used as a control. For xenograft analysis, HMC-1.2 cells (1 x 10⁶) in 50% Matrigel (Corning, 354248, NY, USA) were used. 6 100μL of Head / Serum-free IMDM medium cells were injected into CB-17 SCID mice via sc (subcutaneous, left side).

[0409] The results are shown in Figure 11.

[0410] In the tumor re-challenge analysis, the NN3201 group showed dose-dependent efficacy without additional ADC infusion, whereas the control group resulted in tumor growth. The PoC-DM1 group showed lower tumor growth inhibition than NN3201, indicating that NN3201 had a stronger effect than PoC-DM1.

[0411] Example 10: In vivo pre-toxicity analysis (10 mg / kg)

[0412] An experiment was conducted to evaluate the toxicity of ADC substances by investigating changes in body weight over two weeks after a single high-concentration IV administration to Balb / c mice.

[0413] 7-week-old female BALB / c mice (Daehan Biolink) were acclimated to Specific Pathogen Free (SPF) conditions before use in the experiment. After being randomized into one of six groups (n = 3 / group), mice were administered an ADC vehicle (5 ml / kg, iv tail) and each ADC substance (10 mg / kg, iv tail). The buffer used as the ADC vehicle was a mixture of 20 mM histidine (Merck, 104352), 50 mM NaCl (Sigma, S3014), 5% sucrose (Sigma, S0389) (pH 6.5), and 0.22 μM filter (Merck, S2GPU11RE). Individual doses were calculated based on the animal body weight recorded immediately before administration, with a dose volume of 5 mL / kg body weight (Table 13).

[0414] Group. (NN3201-Abzena ADC) #003-1, #004-2, #003-2 No. of Animals. Volume. 1 Control (vehicle, Abzena-ADC buffer) 3 5 mL / kg 2 #003-1 10 mg / kg 3 3 #004-2 10 mg / kg 3 4 #003-2 10 mg / kg 3

[0415] All animals were observed once daily for mortality, general condition, and clinical signs (time, onset, severity, and recovery). The body weight of all animals was measured daily from the date of grouping. The animals were sacrificed on the 14th day.

[0416] After the experiment was completed, the animals were euthanized (cervical dislocation was performed as soon as possible to minimize pain, as pulmonary congestion may be affected during euthanasia using respiratory anesthetics or a CO2 chamber) and a necropsy was performed to check for the presence of symptoms of pulmonary congestion. The tissue (lungs) was stored in 10% NBF (neutral buffered formalin).

[0417] The results are shown in Fig. 12.

[0418] No abnormal symptoms were observed in any group during the test period.

[0419] During the study period, symptoms of weight abnormalities (more than 5%), such as rapid weight loss or rapid weight gain following administration, were not observed in any group.

[0420] At the end of the experiment (Day 14), compared to the day of administration (Day 0), body weight increased by 0.43 g (2.10%) in the #003-1 10 mg / kg group and by 0.63 g (3.00%) in the #004-2 10 mg / kg group. At the end of the experiment (Day 14), the body weight of the #003-2 10 mg / kg group decreased by 0.27 g (1.29%) compared to the day of administration (Day 0).

[0421] There were no symptoms of pulmonary congestion in the lung tissues of all groups.

[0422] Example 11: In vivo pre-toxicity analysis (20 / 40 / 60 mg / kg)

[0423] An experiment was conducted to evaluate the toxicity of ADC substances by investigating changes in body weight over two weeks after a single high-concentration IV administration to Balb / c mice.

[0424] The analysis was performed in the same manner as in Example 10, except that the groups were assigned as follows (Table 14).

[0425] Group No. of Animals. Volume. Control (Non-treat) 3 - Control (vehicle, ADC buffer) 3 5 mL / kg #003-1 20, 40, or 60 mg / kg 3 #004-2 20, 40, or 60 mg / kg 3

[0426] The results are shown in FIGS. 13a to 13d.

[0427] No abnormal symptoms were observed in the entire group during the test period.

[0428] During the study period, no symptoms of body weight abnormalities (more than 5%), such as rapid weight loss or rapid weight gain following administration, were observed in any group. On the first day after administration (Day 0), weight loss was observed in 0.63 g (3.23%) of the #PoC-DM1 40 mg / kg group and 0.70 g (3.57%) of the #PoC-DM1 60 mg / kg group. However, the lost weight was recovered.

[0429] Although embodiments of the present invention have been described above, those skilled in the art may modify and change the present invention in various ways by adding, changing, deleting, or adding components, etc., without departing from the spirit of the invention as described in the claims, and such modifications and changes are also to be included within the scope of the rights of the present invention.

Claims

Claim 1 Antibody-drug conjugate of Formula 1 below: <Formula 1>Ab-(L) x -(D) y In the above Chemical Formula 1, Ab is an anti-c-Kit antibody that specifically binds to c-Kit or its antigen-binding fragment comprising heavy chain CDR1 of SEQ ID NO. 1, heavy chain CDR2 of SEQ ID NO. 2, heavy chain CDR3 of SEQ ID NO. 3, light chain CDR1 of SEQ ID NO. 4, light chain CDR2 of SEQ ID NO. 5, and light chain CDR3 of SEQ ID NO. 6; D is monomethylauristatin E (MMAE); x is an integer from 1 to 8; y is an integer from 1 to 8; and L has the structure of the following Chemical Formula 2; <Chemical Formula 2> (L1) m -(S) n -(L2) p In the above chemical formula 2, L1 is a linker connecting Ab and S or Ab and L2, , , , , , , , , , , , , , , , , , , , , , , , , , , , , or And, S is a spacer in a form in which a polymer is bonded or not bonded, , , , and The polymer is selected from the group consisting of polyalkylene, polyalkylene glycol, polyvinylpyrrolidone, polyacrylate, polyoxazoline, polyvinyl alcohol, polyacrylamide or polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(orthoester), polycarbonate, poly(iminocarbonate), polyamide, copolymer of divinyl ether-maleic anhydride or styrene-maleic anhydride, polysaccharide, or polyglutamic acid, and the spacer in a form to which the polymer is bonded or not bonded may include or not include aspartate, glutamate, or a combination thereof; L2 is A cleavable linker selected from the group consisting of valine-citrulline-p-aminobenzyl carbamoyl (Val-Cit-PAB), alanine-phenylalanine-p-aminobenzyl carbamoyl (Ala-Phe-PAB), alanine-alanine-p-aminobenzyl carbamoyl (Ala-Ala-PAB), valine-alanine-p-aminobenzyl carbamoyl (Val-Ala-PAB), phenylalanine-lysine-p-aminobenzyl carbamoyl (Phe-Lys-PAB), alanine-alanine-glycine-p-aminobenzyl carbamoyl (Ala-Ala-Gly-PAB) and glycine-glycine-glycine-p-aminobenzyl carbamoyl (Gly-Gly-Gly-PAB) linkers; m is an integer from 0 to 8; and n is 0 to 8 p is an integer from 1 to 8. Claim 2 An antibody-drug conjugate according to claim 1, wherein the antibody or its antigen-binding fragment specifically binds to one or more of R122, Y125, R181, K203, R205, S261 and H263 in SEQ ID NO.

12. Claim 3 delete Claim 4 delete Claim 5 An antibody-drug conjugate according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable domain represented by SEQ ID NO. 7 and a light chain variable domain represented by SEQ ID NO.

8. Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 An antibody-drug conjugate according to claim 1, characterized in that m is 1 to 4, n is 1 to 4, and p is 1 to 4. Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 An antibody-drug conjugate according to claim 1, wherein L comprises a linker selected from the group consisting of the following chemical formulas 3 to 5. <Chemical Formula 3> ,<Chemical Formula 4> or <Chemical Formula 5> Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 The antibody-drug conjugate of claim 1, characterized in that the antibody-drug conjugate is selected from the group consisting of the following chemical formulas 6 and 7: <Chemical Formula 6> and <Chemical Formula 7> In the above formula, a is an integer from 1 to 8, and b is an integer from 1 to 4. Claim 22 Antibody-drug conjugate selected from the group consisting of the following chemical formulas 6 to 8: <Chemical Formula 6> , <Chemical Formula 7> and <Chemical Formula 8> , in the above formula, Ab is an anti-c-Kit antibody that specifically binds to c-Kit or its antigen-binding fragment comprising heavy chain CDR1 of SEQ ID NO. 1, heavy chain CDR2 of SEQ ID NO. 2, heavy chain CDR3 of SEQ ID NO. 3, light chain CDR1 of SEQ ID NO. 4, light chain CDR2 of SEQ ID NO. 5, and light chain CDR3 of SEQ ID NO. 6; a is an integer from 1 to 8, and b and c are each integers from 1 to 4. Claim 23 A pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate of claim 1 or claim 22. Claim 24 delete Claim 25 delete

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