Antibody against c-kit and use thereof

Abstract

Disclosed are an antibody against c-Kit and the use thereof, in particular, an antibody that binds to c-Kit or an antigen-binding fragment thereof, a nucleic acid comprising a nucleotide sequence encoding the antibody, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector. Further disclosed are a pharmaceutical composition, conjugate and chimeric antigen receptor comprising the antibody, and a treatment method using same.

Description

Antibodies against c-Kit and uses thereof TECHNICAL FIELD

[0001] The present invention relates to antibodies binding to c-Kit and uses thereof, in particular in the treatment of allergic diseases. BACKGROUND

[0002] Allergic diseases are defined by the World Allergy Organization and WHO as the sixth largest chronic disease in the world, among which urticaria is one of the most common allergic diseases, and chronic urticaria is a very important type. According to statistics, the incidence of chronic urticaria in Asia is about 1.4%, higher than that in Europe (0.5%) and North America (0.1%) (ZT Zhao et al., China Medical News. 2020, 35(22)). Its main manifestations are recurrent wheals and / or angioedema, usually accompanied by severe itching or burning sensation and other subjective discomfort symptoms, which seriously affect the work and study of patients, the quality of life and mental health, and cause heavy social and economic burden. The first-line treatment currently uses second-generation antihistamines, but the effective rate of chronic spontaneous urticaria patients is less than 50%, and after increasing the dose of second-line treatment, the response rate is improved, but 1 / 3 to 1 / 4 of the patients still have poor improvement of symptoms (Fok JS et al., Allergy. 2021 Oct; 76(10): 2965-2981), and there is still a lack of more rapid and effective drug treatment for chronic urticaria in clinical practice.

[0003] Mast cells are immune response effector cells, present in many places in human tissues, especially in places where the human body is in contact with the external environment, such as the intestinal tract and the skin (Arthur G et al., Clinical Implications. Chest. 2016; 150(3): 680-693.). Mast cells are mainly involved in host defense, immune regulation, allergic reactions and chronic inflammation by secreting bioactive products with a wide range of properties, which are preformed and stored in their cytoplasmic granules, including proteoglycans, lysosomal enzymes, biogenic amines, cytokines and growth factors (Gilfillan AM et al., Adv Exp Med Biol. 2011; 716: 2-12.). Abnormal activation of mast cells is involved in the pathological process of allergic diseases such as asthma, urticaria and mastocytosis (Levi-Schaffer F et al., J Allergy Clin Immunol. 2022; 149(6): 1833-1844.). Skin mast cells are the main effector cells in the pathological process of urticaria, and the number of mast cells increases in the skin lesions and non-lesion areas of chronic spontaneous urticaria and chronic inducible urticaria (Kolkhir P et al., J Allergy Clin Immunol. 2022; 149(6): 1819-1831).

[0004] Mast cell precursors and mast cells both highly express c-Kit, which belongs to the third receptor tyrosine kinase family and can also be called CD117. Stem cell factor (SCF) is the only ligand of c-Kit. SCF / c-kit plays an important role in the development, function, migration and survival of mast cells. In addition, SCF / c-Kit plays an important role in hematopoiesis, spermatogenesis, and melanogenesis, and mice with c-Kit sub-effect mutations severely lack mast cells and show anemia, infertility, and impaired melanogenesis (Jing Chen et al., Blood, 2009; 114(22): 2542; Waskow C et al., Adv Exp Med Biol. 2002; 512: 1-10.).

[0005] There are many clinical stages and marketed products targeting c-Kit. At present, small molecule inhibitors are mainly used, and the main indications are gastrointestinal stromal tumors and systemic mastocytosis. Among them, the anti-c-Kit antibody Barzolvolimab is mainly used for chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CIndU), and the current phase II clinical study has reached the main endpoint. Subsequent clinical studies are also being carried out in nodular prurigo. The results of its phase II clinical study showed that 75 mg / person, 150 mg / person once every four weeks or 300 mg / person once every eight weeks, all significantly improved the symptoms of urticaria, the severity of pruritus score [ISS7] and the severity of urticaria score [HSS7] were significantly improved, the response was rapid after initial administration, and the efficacy was long-lasting, but the most common AEs included neutropenia and color change. The clinical data of Barzolvolimab suggest that inhibiting c-Kit activity can effectively improve urticaria. SUMMARY

[0006] The present application provides a c-Kit antibody, which has higher affinity than Barzolvolimab, more effectively inhibits SCF-induced cell proliferation, cytokine release and degranulation activity caused by IgE crosslinking, and completely removes the interaction with Fc gamma receptor and C1q after sequence optimization, avoiding the safety risks brought by Fc effector function.

[0007] Correspondingly, in one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to c-Kit, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0008] (1) the VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 4, 46, 6, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 51, 10, 11, respectively; or

[0009] (2) the VH comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 31, 56, 25, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 61, 10, 34, respectively; or

[0010] (3) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, 6, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 9, 10, 11, respectively; or

[0011] (4) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 14, 15, 16, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, 21, respectively; or

[0012] (5) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 24, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 9, 10, 28, respectively; or

[0013] (6) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 31, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 9, 10, 34, respectively; or

[0014] (7) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 37, 38, 39, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 42, 10, 43, respectively.

[0015] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49.

[0016] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59.

[0017] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0018] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17.

[0019] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0020] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.

[0021] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0022] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 44, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0023] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 54, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 59.

[0024] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0025] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 17.

[0026] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 22, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 26.

[0027] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 29, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 32.

[0028] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40.

[0029] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0030] In some embodiments, the antibody belongs to an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0031] In some embodiments, the antibody belongs to a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

[0032] In some embodiments, the antibody comprises:

[0033] (i) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52; or

[0034] (ii) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62.

[0035] In some embodiments, the antibody comprises:

[0036] (i) a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 47, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 52; or

[0037] (ii) a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 57, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 62.

[0038] In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab, Fab’, F(ab’)2, Fv, scFv, and ds-scFv.

[0039] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0040] In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen.

[0041] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof disclosed herein.

[0042] In some embodiments, the nucleic acid comprises:

[0043] (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 45, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 50; or

[0044] (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 55, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 60; or

[0045] (3) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 3, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8; or

[0046] (4) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18; or

[0047] (5) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27; or

[0048] (6) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 30, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33; or

[0049] (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53; or

[0050] In some embodiments, the nucleic acid comprises:

[0051] (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53; or

[0052] (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63.

[0053] In yet another aspect, the present disclosure provides a vector comprising the nucleic acid disclosed herein.

[0054] In yet another aspect, the present disclosure provides a host cell comprising the nucleic acid or vector disclosed herein.

[0055] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (i) the antibody or antigen binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0056] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0057] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0058] In yet another aspect, the present disclosure provides a conjugate comprising the antibody or antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

[0059] In yet another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antibody or antigen binding fragment thereof disclosed herein.

[0060] In yet another aspect, the present disclosure provides a method for treating a disease in a subject comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a CAR disclosed herein.

[0061] In yet another aspect, the present disclosure provides an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a CAR disclosed herein for use in treating a disease in a subject.

[0062] In yet another aspect, the present disclosure provides use of an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a CAR disclosed herein in the manufacture of a medicament for treating a disease in a subject.

[0063] In some embodiments of any of the above aspects, the disease is a disease associated with c-Kit expression, for example, an allergic disease, gastrointestinal stromal tumor, systemic mastocytosis, eosinophilic esophagitis, sickle cell disease, hematopoietic system disease, severe combined immunodeficiency, myelodysplastic syndrome, chronic granulomatous disease, diabetic macular edema, wet age-related macular degeneration.

[0064] In some embodiments, the allergic disease is selected from allergic dermatitis (e.g., urticaria, eczema, nodular prurigo, contact dermatitis, etc.), allergic rhinitis, allergic asthma, allergic purpura, anaphylactic shock, allergic conjunctivitis. In preferred embodiments, the allergic disease is selected from urticaria, nodular prurigo, asthma.

[0065] In some embodiments, the urticaria is chronic urticaria, for example, chronic spontaneous urticaria or chronic inducible urticaria.

[0066] In some embodiments, the method or use further comprises administering to the subject a second therapeutic agent.

[0067] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 shows the binding affinity of anti-c-Kit chimeric antibodies to human and cynomolgus monkey c-Kit HEK293 stable cell lines; A and B: binding affinity of anti-c-Kit chimeric antibodies to human c-Kit HEK293 stable cell lines, C and D: binding affinity of anti-c-Kit chimeric antibodies to cynomolgus monkey c-Kit HEK293 stable cell lines.

[0069] Figure 2 shows the results of anti-c-Kit chimeric antibody inhibiting phosphorylation of human c-Kit HEK293 stable cell line induced by stem cell growth factor (SCF).

[0070] Figure 3 shows the results of anti-c-Kit chimeric antibody inhibiting SCF-induced M-07e cell proliferation.

[0071] Figure 4 shows the binding affinity of anti-c-Kit humanized antibody to human and cynomolgus monkey c-Kit HEK293 stable cell line.

[0072] Figure 5 shows the results of anti-c-Kit humanized antibody inhibiting SCF-induced M-07e cell proliferation.

[0073] Figure 6 shows the results of anti-c-Kit humanized antibody inhibiting mast cell degranulation.

[0074] Figure 7 shows the results of anti-c-Kit humanized antibody inhibiting SCF-induced mast cell cytokine release.

[0075] Figure 8 shows the results of pharmacokinetic curve and half-life of anti-c-Kit humanized antibody h67F1 in mice.

[0076] Figure 9 shows the results of body temperature of mice in each group changing with time after DNP-HAS challenge.

[0077] Figure 10 shows the results of clinical scores of mice in each group after DNP-HAS challenge.

[0078] Figure 11 shows the results of body temperature of mice in each group changing with time after DNP-HAS challenge. DETAILED DESCRIPTION

[0079] The above features and advantages of the present application, and additional features and advantages thereof, will be more clearly understood from consideration of the following detailed description when taken in conjunction with the accompanying drawings.

[0080] The embodiments described herein with reference to the accompanying drawings are explanatory, illustrative, and serve the purpose of general understanding of the present application. The embodiments should not be interpreted as limiting the scope of the present application. The same or similar elements and elements having the same or similar functions are denoted by the same reference numerals throughout the specification.

[0081] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and phrases related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and molecular genetics employed herein conform to standard usage, unless otherwise indicated. Also, the definitions and explanations of the relevant terms are provided below for better understanding of the present application.

[0082] Definitions

[0083] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "the antibody" includes multiple antibodies, and so forth.

[0084] The term "comprising" and variations thereof as used herein are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0085] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, for example, the composition comprises A and / or B can be interpreted to mean that the composition comprises A alone, B alone, or A and B in combination.

[0086] As used herein, the term "about" generally means within 0.5% - 10% above or below the indicated numerical value, e.g., within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the indicated numerical value.

[0087] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Such molecules are typically comprised of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the antibody heavy and light chains contain the binding sites for the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation).

[0088] The heavy chain of an immunoglobulin can be divided into three functional regions: an Fd region, a hinge region, and an Fc region (fragment crystallizable). The Fd region comprises the VH and CH1 domains and, in combination with the light chain, forms the Fab (antigen-binding fragment) that is responsible for antigen binding. The Fc fragment is responsible for the effector functions of the immunoglobulin, including, for example, complement fixation and binding to cognate Fc receptors on effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portions to move freely in space relative to the Fc region. The hinge domain is structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.

[0089] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions structurally and functionally: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes amino acids from the carboxy-terminal end of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the flexibility of the fragment of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues in the CH2 domain. Structural and flexible allowed conformational changes in the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the Fc portion of the antibody.

[0090] A “light chain variable region” (VL) or “heavy chain variable region” (VH) is composed of “framework” regions separated by three “complementarity determining regions” or “CDRs.” The framework regions serve to align the CDRs that specifically bind to an epitope of an antigen. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains comprise, from amino-terminus to carboxy-terminus, the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0091] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure can use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined according to the Kabat definition.

[0092] Based on the amino acid sequences of the constant regions of the heavy chains of the antibodies, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and the like. Based on the amino acid sequences of the light chains, the light chains of the antibodies can be assigned to lambda (l) chains and kappa (K) chains.

[0093] As used herein, the term "antibody" shall be construed in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multi-specific antibodies (e.g., bi-specific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0094] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, each antibody in the population is identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology, nor is it intended to be limited to antibodies produced in any particular manner.

[0095] The term "bispecific antibody" is understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of one target. The term "bispecific antibody" as used herein is to be understood in its broadest sense, including full-length bispecific antibodies and antigen binding fragments thereof. Bispecific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification of the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0096] The term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody elicits a reduced likelihood of adverse immune reactions in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0097] The term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., murine antibody) are replaced with corresponding amino acids from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids can also be permissible, as long as they still retain the ability of the antibody to bind to a particular antigen. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody comprising sequences derived from non-human immunoglobulin. In some cases, CDR region residues in the human immunoglobulin (recipient antibody) are replaced by CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that have the desired properties, affinities, and / or capabilities. In some cases, FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody can contain amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0098] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

[0099] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, this term also includes "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form the antigen binding region, as well as modified forms of any of the foregoing fragments that retain antigen binding

[0100] As used herein, the term "binds" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0101] Affinity is a measure of the strength of binding between an antibody and the associated antigen. Affinity involves both the avidity between the antigenic determinant and the antigen binding site of the antibody and the number of associated binding sites present on the antibody. Typically, an antibody will bind an antigen with a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or has a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 . It is generally accepted that any K -4 M value greater than 10 D M indicates non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and different variations thereof known in the art.

[0102] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein or proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually include at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimum binding site of an antibody and are therefore the specific targets of an antibody or antigen binding fragment thereof.

[0103] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions when aligned. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0104] Furthermore, in determining the extent of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions that replace an amino acid residue with another amino acid residue having similar chemical structure that have little or substantially no effect on the function, activity or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0105] Such conservative substitutions are preferably substitutions of one amino acid by another amino acid residue in one of the following groups (a) to (e): (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, lie, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0106] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin or to Glu; Met to Leu, to Tyr or to lie; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie or to Leu.

[0107] The term "vector" as used herein is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and of which it is capable of replicating itself in suitable host cells, transferring the inserted nucleic acid molecule into and / or between host cells.

[0108] The term "host cell" as used herein refers to a cell into which an expression vector has been introduced.

[0109] The term "pharmaceutically acceptable" means that which the carrier or excipient is compatible with the other ingredients of the composition and not deleterious or substantially deleterious to the recipient thereof, and / or such carrier or excipient is approved or approvable by a regulatory agency of the Federal or a state government of the United States or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for inclusion in a drug product for parenteral administration to humans.

[0110] As used herein, the terms "treatment," "therapy," "treatment," and the like refer to the application of an agent or performance of a procedure for the purpose of effecting an outcome. These effects can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or therapeutic in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the symptoms of the disease to regress. Treatment can refer to any measure of success in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as a decrease in symptoms; remission; elimination of disease symptoms or disease conditions that are easier for the patient to tolerate; slowing in rate of decline or deterioration; or an increase in the last node of deterioration. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including results of physician examination. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate, or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0111] As used herein, the term "effective amount" refers to an amount of an agent administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0112] As used herein, the term "subject" refers to any mammalian subject in which diagnosis, treatment, or therapy is desired. A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0113] Anti-c-Kit antibodies

[0114] The present disclosure provides an antibody or antigen-binding fragment thereof that binds c-Kit, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0115] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 4, 46, 6, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 51, 10, 11, respectively.

[0116] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 31, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 9, 10, 34, respectively.

[0117] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 4, 5, 6, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 9, 10, 11, respectively.

[0118] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 14, 15, 16, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 19, 20, 21, respectively.

[0119] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 24, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 9, 10, 28, respectively.

[0120] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 31, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 9, 10, 34, respectively.

[0121] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 37, 38, 39, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, each having an amino acid sequence as set forth in SEQ ID NOs: 42, 10, 43, respectively.

[0122] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49.

[0123] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59.

[0124] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0125] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17.

[0126] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0127] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.

[0128] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0129] In some embodiments, the VH comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to c-Kit. In some embodiments, the VL comprises a functional variant of an amino acid sequence as disclosed herein above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to c-Kit.

[0130] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0131] In the context of a functional variant, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is between 1 and 33%, more preferably is between 5 and 30%, more preferably is between 10 and 25%, more preferably is between 15 and 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be between 1 and 20, preferably between 1 and 10, more preferably between 1 and 7, still more preferably between 1 and 5, most preferably between 1 and 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0132] In some embodiments, the insertion, deletion, and / or substitution can be made in a framework (FR) region, e.g., in FR1, FR2, FR3, and / or FR4.

[0133] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid for another within a group (a) to (e) below: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0134] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to He, or to Leu.

[0135] In preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 44, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0136] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 54, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 59.

[0137] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0138] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 17.

[0139] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 22, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 26.

[0140] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 29, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 32.

[0141] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 35, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 40.

[0142] In some embodiments, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody. In preferred embodiments, the antibody is a humanized antibody.

[0143] Based on the amino acid sequences of the constant regions of the heavy chains, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to either lambda (l) chain or kappa (K) chain. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0144] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subclass selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgGl antibody.

[0145] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52.

[0146] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62.

[0147] In some embodiments, the heavy chain and / or light chain comprises a functional variant of an amino acid sequence as disclosed herein above formed by insertion, deletion and / or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to c-Kit.

[0148] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0149] In the context of a functional variant, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is between 1 and 33%, more preferably is between 5 and 30%, more preferably is between 10 and 25%, more preferably is between 15 and 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be between 1 and 20, preferably between 1 and 10, more preferably between 1 and 7, still more preferably between 1 and 5, most preferably between 1 and 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0150] In some embodiments, the insertion, deletion and / or substitution can be made in a constant region (e.g., CH1, CH2, CH3, and / or CL) and / or a framework (FR) region (e.g., FR1, FR2, FR3, and / or FR4).

[0151] In preferred embodiments, the antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 47, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 52; or

[0152] In other preferred embodiments, the antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 57, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 62.

[0153] The antibodies disclosed herein can be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to c-Kit. Examples of antigen-binding fragments include, but are not limited to: Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen binding activity.

[0154] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In another preferred embodiment, the antigen-binding fragment is scFv.

[0155] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen.

[0156] The antibodies disclosed herein can comprise an Fc region. The Fc region can be of any isotype, including but not limited to IgGl, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is or is derived from an IgGl or IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG4 Fc. When the Fc region is a human IgG4 Fc, it can comprise a S228P mutation to enhance the stability of the IgG4 antibody. Thus, in some embodiments, the Fc region is a human IgG4 Fc comprising a S228P mutation.

[0157] In some embodiments, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC, and / or Clq, Fcy receptor binding. For example, the Fc region can be of the IgG4 isotype, or of a non-IgG4 type, e.g., IgGl, IgG2, or IgG3, that has been mutated such that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, e.g., in Dall’Acqua WF, et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, an IgGl Fc region can comprise an amino acid sequence having one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. An IgG4 Fc region can comprise an amino acid sequence having one or more of the following amino acid substitutions compared to the wild-type sequence: S228P, E233P, F234A, L235A, L235E, G236del, G237A, P238S, D265A, P329A, A330R, and R409K. In preferred embodiments, the IgG4 Fc region comprises S228P, L235E, and P329A (the SPLEPA mutation).

[0158] In other embodiments, the Fc region has an extended serum half-life. For examples of altering (e.g., decreasing or increasing) the in vivo half-life of an antibody, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patent Nos. 5,869,046; 6,121,022; 6,277,375; and 6,165,745, all of which are incorporated herein by reference in their entireties. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to decrease the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge region-Fc domain fragment) to increase the in vivo half-life of the antibody. In a particular embodiment, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. For example, the Fc region can comprise an amino acid sequence having one or more of the following amino acid substitutions compared to the wild-type sequence: T250Q, T250E, M252Y, S254T, T256E, E258F, T307Q, T307A, V308F, Q311V, E380A, V427T, M428L, M428F, N434S, N434A, N434H, and N434F. In some embodiments, the Fc region comprises M252Y, S254T, and T256E (YTE mutations). In some embodiments, the Fc region comprises M428L and N434A (LA mutations).

[0159] Nucleic acid

[0160] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein.

[0161] The term "nucleic acid" includes single- and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phenylphosphonothioate, phenylphosphonate, and phosphoramidate.

[0162] In some embodiments, the present application provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0163] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41.

[0164] In some embodiments, the present application provides a nucleic acid molecule encoding any one of the heavy chains disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the heavy chains disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding any one of the light chains disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the light chains disclosed herein.

[0165] In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53. In some embodiments, the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63.

[0166] In some embodiments, the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In some embodiments, the present application provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present application provides a deoxyribonucleic acid (DNA) comprising a deoxyribonucleotide sequence encoding an antibody disclosed herein.

[0167] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into a human cell in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is comprised in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is integrated into the genome of a cell.

[0168] In some embodiments, a ribonucleic acid (RNA) can be introduced into a human cell in vivo. In some embodiments, a ribonucleic acid (RNA) of the present application is contained in a vector or delivery agent.

[0169] Vectors

[0170] The present disclosure provides vectors comprising the nucleic acids disclosed herein.

[0171] In some embodiments, a vector is an expression vector capable of expressing a polypeptide comprising a heavy chain or light chain variable region of an antibody. For example, the present application provides an expression vector comprising any of the nucleic acid molecules described above.

[0172] Any vector can be suitable for use in the present disclosure. In some embodiments, a vector is a viral vector. In some embodiments, a vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large T cDNA, pBABE-hygro-hTERT, pMKO.lGFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0173] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as lambda GT10, lambda GT11, lambda Zap II (Stratagene), lambda EMBL4, and lambda NM1149 can also be used. Examples of plant expression vectors that can be used in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0174] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain the functions of a replication system in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, COLEL, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0175] For example, the vector can be an adenoviral vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered into a subject, then in vivo into a cell of the subject, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

[0176] Host cells

[0177] The present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0178] Any cell can be used as a host cell for the nucleic acids or vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0179] The host cells of the present application are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present application can be administered to a subject in vivo, and the host cells express the antibodies disclosed herein in vivo.

[0180] The present application provides host cells into which any of the above-described vectors have been introduced. The present application also provides methods of making the antibodies of the present application, comprising a) culturing the host cells disclosed herein under conditions suitable for production of the antibodies; and b) obtaining the antibodies from the culture.

[0181] Pharmaceutical Compositions

[0182] The present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0183] The antibodies or antigen-binding fragments thereof of the present application (also referred to herein as“active compounds”) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise the antibody or antigen-binding fragment thereof, together with a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such vehicles and agents for use in

[0184] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0185] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0186] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0187] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0188] Oral composition generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash in which the compound is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0189] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide.

[0190] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into

[0191] The active compounds can also be prepared in the form of suppositories (e.g., using conventional suppository

[0192] In one embodiment, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0193] The present application provides therapeutic compositions comprising the antibodies or antigen binding fragments thereof of the present application. The therapeutic compositions according to the present application will be administered with suitable carriers, excipients and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions with

[0194] Conjugates

[0195] The present disclosure provides conjugates comprising the antibodies or antigen binding fragments thereof disclosed herein, and a chemical moiety conjugated thereto.

[0196] In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC."

[0197] The term "conjugate" or "linkage" can refer to joining two polypeptides into one continuous polypeptide molecule. In one embodiment, the antibody is linked to a chemical moiety. In another embodiment, the antibody linked to a chemical moiety is further linked to a lipid or other molecule to the protein or peptide to increase its half-life in vivo. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, in which a reaction between the antibody moiety and the chemical moiety results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the chemical moiety.

[0198] The chemical moiety can be linked to the antibody of the present application using any number of means known to those of skill in the art. Both covalent and non-covalent means of attachment can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with a suitable functional group on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of a number of known linker molecules. The linker can be any molecule that serves to link the antibody to the chemical moiety. The linker is capable of forming a covalent bond to both the antibody and the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0199] In certain cases, it is desirable to release the chemical moiety from the antibody when the immunoconjugate reaches its target site. Thus, in these cases, the immunoconjugate will comprise a linkage that is cleavable in the vicinity of the target site.

[0200] The conditions experienced by the enzymatic activity or immunoconjugate within the target cell or in the vicinity of the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0201] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of skill in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0202] Antibodies disclosed herein can be derivatized or linked to another molecule, such as another peptide or protein. Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detector agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0203] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different type). Suitable cross-linking agents include heterobifunctional or homobifunctional cross-linking agents having two distinct reactive moieties separated by a suitable spacer region (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or a homobifunctional cross-linking agent (such as disuccinimidyl suberate). Such linkers are commercially available.

[0204] In some embodiments of the conjugates disclosed herein, the chemical moiety can be, for example, a therapeutic agent and a detectable moiety.

[0205] In some embodiments, the therapeutic agent can be any therapeutic agent useful in the treatment of allergic diseases, such as an antihistamine or a hormone.

[0206] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0207] Antibodies can be conjugated to detectable labels; for example, detectable labels that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used.

[0208] Antibodies or antigen-binding fragments can also be conjugated to enzymes that are useful for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When antibodies or antigen-binding fragments are conjugated to detectable enzymes, detection can be achieved by the addition of additional reagents that the enzyme uses to produce a recognizable reaction product. For example, when horseradish peroxidase reagents are present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0209] Antibodies can be fused to self-labeling protein tags, such as HaloTag. For example, the protein tag can be cloned to the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0210] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0211] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with predetermined polypeptide epitopes recognized by a second reporter group (such as a leucine zipper pair sequence, a binding site for biotin, a metal binding domain, an epitope tag) that are recognized by a second reporter group. In some embodiments, the tag is attached by a spacer arm of various lengths to reduce potential steric hindrance.

[0212] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of a target antigen by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I.

[0213] Chimeric antigen receptor

[0214] The present disclosure provides chimeric antigen receptors (CARs) comprising an antibody or antigen-binding fragment thereof disclosed herein. The present disclosure also provides genetically modified cells comprising a chimeric antigen receptor disclosed herein.

[0215] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule engineered to contain an antigen-binding domain that targets a specific antigen and, upon binding to that antigen, activates an immune cell (e.g., a T cell or NK cell, such as a naive T cell, central memory T cell, effector memory T cell, or a combination thereof) to attack and destroy cells bearing that antigen. When these antigens are present on target cells, the CAR-expressing immune cells can target and kill the target cells.

[0216] A classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain. The antigen-binding domain is typically an antigen-binding fragment derived from a monoclonal antibody (mAb) (e.g., scFv), but it can be based on other formats containing antibody-like antigen-binding sites or antigen-binding domains derived from natural ligands of the antigen. A hinge domain is often required to separate the antigen-binding domain from the membrane and allow for its proper orientation. A common hinge domain used is the Fc of IgGl. Depending on the antigen, more compact spacers can suffice, such as the stalk from CD8a, and even just the IgGl hinge alone. A transmembrane domain anchors the protein in the cell membrane and links the hinge domain to the intracellular domain (endodomain).

[0217] According to at least one non-limiting view, there have been at least three "generations" of CAR molecules. In the first generation CARs, they were designed to have an intracellular domain derived from the intracellular portion of the gamma chain of Fc epsilon Rl or CD3 zeta. Thus, these first generation CARs transmit immune signal 1, which is sufficient to trigger T cell killing of cognate target cells, but is not able to fully activate T cell proliferation and survival. To overcome this limitation, second generation CARs have been constructed, which have a composite intracellular domain generated by fusing the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3 zeta, so that both an activation signal and a costimulatory signal can be transmitted upon antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal, immune signal 2, which triggers T cell proliferation. Some CARs have also been described that include TNF receptor family intracellular domains, such as the closely related OX40 and 41BB, which transmit survival signals. Even more potent third generation CARs have now been described, which have intracellular domains capable of transmitting activation, proliferation, and survival signals.

[0218] Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more costimulatory domains.

[0219] An "antigen binding domain" refers to the portion of a chimeric antigen receptor that recognizes an antigen. In classic CARs, the antigen binding domain comprises a single chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been created with domain antibodies (dAbs), VHH antigen binding domains, or antigen binding domains derived from natural ligands of the antigen. In the present application, the antigen binding domain can be an antibody or antigen binding fragment thereof as described herein.

[0220] A "hinge domain" positions the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation to function. CARs optionally include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise an amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular region of type 1 membrane proteins such as CD8 (e.g., CD8a), CD4, CD28, 4-1BB, and CD7, which can be from the wild-type hinge region of these molecules or can be altered.

[0221] As used herein, a“transmembrane domain” (TM domain) refers to a portion of a CAR that, optionally via a hinge domain, fuses an extracellular binding moiety with intracellular moieties (e.g., a costimulatory domain and an intracellular signaling domain) and anchors the CAR to the plasma membrane of an immune effector cell. A transmembrane domain is typically a hydrophobic region of a CAR that spans the plasma membrane. The TM domain can be a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein) or a fragment thereof, an artificial hydrophobic sequence, or a combination thereof.

[0222] An intracellular domain (endodomain) is the signaling portion of a chimeric antigen receptor. It contains a signaling domain and one or more costimulatory domains. Upon antigen recognition, the receptor clusters naturally CD45 and CD148 away from the synapse and signals to the cell, thereby activating one or more immune cell effector functions (e.g., a native immune cell effector function). The most commonly used endodomain component is the endodomain component of CD3 zeta, which contains 3 ITAMs. Upon antigen binding, it transmits an activation signal to the T cell. CD3 zeta can not provide a fully sufficient activation signal and can require additional costimulatory signaling. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).

[0223] An“intracellular signaling domain” refers to a portion of a CAR polypeptide that participates in transducing the information that the CAR is bound to a target antigen to the interior of an immune effector cell to elicit effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to a target cell to which the CAR is bound, or other cellular responses elicited upon binding of an antigen to an extracellular CAR domain. Non-limiting examples of intracellular signaling domains containing immunoreceptor tyrosine-based activation motifs (ITAMs) include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 delta, CD3 eta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0224] A“costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for effective activation and function of T lymphocytes upon binding of antigen. The costimulatory domain can be a costimulatory domain of, e.g., 4-1BB, CD27, CD28, or OX40.

[0225] Therapeutic methods and uses

[0226] The present disclosure provides methods for treating a disease in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein.

[0227] The present disclosure also provides the use of an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein in the manufacture of a medicament for treating a disease in a subject.

[0228] The present disclosure also provides an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, a conjugate disclosed herein, or a chimeric antigen receptor disclosed herein for use in treating a disease in a subject.

[0229] In some embodiments, the disease is a disease associated with c-Kit expression or a c-Kit positive disease. For example, the disease is a mast cell associated disease. A mast cell associated disease refers to a condition in which mast cell activity causes pathology and / or mast cells are present in abnormal amounts (e.g., higher than normal amounts or lower than normal amounts) in different parts of the body. For example, a mast cell associated condition can show pathological mast cell accumulation in any or all organs and tissues possible and / or abnormal release of one or more mast cell mediators (e.g., inflammatory mediators). Non-limiting examples of inflammatory mediators released by mast cells include any of the following: (i) granule-associated mediators including histamine, serotonin, and various proteases and peptidases; (ii) eicosanoids such as prostaglandin D2 (PGD2) and leukotriene C4 (LTC4); and (iii) cytokines including interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNFa) and chemokines including CCL-2, CCL-3, CCL-5, and CXCL8.

[0230] In some embodiments, the disease includes, but is not limited to, an allergic disease, gastrointestinal stromal tumor, systemic mastocytosis, eosinophilic esophagitis, sickle cell disease, hematopoietic system disease, severe combined immunodeficiency, myelodysplastic syndrome, chronic granulomatous disease, diabetic macular edema, wet age-related macular degeneration, and the like. The allergic disease can include, but is not limited to, allergic dermatitis (e.g., urticaria, eczema, nodular prurigo, contact dermatitis, and the like), allergic rhinitis, allergic asthma, allergic purpura, anaphylactic shock, allergic conjunctivitis. In some embodiments, the disease is an allergic disease. The allergic disease can be selected from, for example, urticaria, nodular prurigo, asthma. The urticaria can include chronic urticaria, for example, chronic spontaneous urticaria or chronic inducible urticaria.

[0231] In some embodiments, the dosage administered to a subject can vary depending on the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount of the antibody to administer to a human or other subject to treat a medical condition. The precise amount required can depend on many factors such as the activity of the antibody and the route of administration.

[0232] A dose of an antibody, composition, conjugate, or chimeric antigen receptor described herein can be administered to a mammal in a suitable period of time either as a single dose or in a series of sub-doses, e.g., as needed, daily, semi-weekly, weekly, biweekly, semi-monthly, bimonthly, semi-annually, or annually. Dose units comprising an effective amount of an antibody, composition, conjugate, or chimeric antigen receptor can be administered as a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily as needed.

[0233] A suitable mode of administration can be selected by a physician. The route of administration can be parenteral administration, e.g., administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, conjugate, or chimeric antigen receptor is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, e.g., orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0234] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the antibody or antigen-binding fragment thereof, pharmaceutical composition, conjugate, or chimeric antigen receptor disclosed herein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In certain embodiments, the antibody, composition, conjugate, or chimeric antigen receptor disclosed herein is administered prior to, substantially simultaneously with, or following administration of the second therapeutic agent.

[0235] Kit / administration device

[0236] The present disclosure provides a kit or administration device comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a host cell disclosed herein, a conjugate disclosed herein, a pharmaceutical composition disclosed herein, or a chimeric antigen receptor disclosed herein.

[0237] In some embodiments, the kit or administration device comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions as set out herein, e.g., an antibody or antigen-binding fragment disclosed herein.

[0238] In particular embodiments, the kit comprises a first container comprising an antibody disclosed herein. In particular embodiments, the kit comprises a first container, which is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit further comprises a second container containing a pharmaceutically acceptable fluid.

[0239] In particular embodiments, provided herein are injection devices comprising an antibody. In particular embodiments, the injection device comprises an antibody in a sterile solution. In particular embodiments, the injection device is a syringe.

[0240] In one embodiment, the kit includes instructional materials that disclose the manner in which the antibodies of the disclosure are used. The instructional materials can be written, in electronic form (such as computer diskette or CD-ROM), or in visual form (such as video files). The kit can further include additional components to facilitate the application for which the kit is designed. Thus, for example, the kit can additionally contain tools for detection of a label (e.g., enzyme substrates for enzymatic labels, filter sets for detection of fluorescent labels, appropriate secondary labels such as a second antibody, etc.). The kit can also include buffers and other reagents commonly used in practicing the particular method. Such kits and suitable contents are well known to those of skill in the art.

[0241] Examples

[0242] The following examples are given for the purpose of illustrating various embodiments of the application and are not meant to limit the present application in any way. This example, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the application. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the application as defined by the scope of the claims.

[0243] Example 1 Generation of Anti-c-Kit Chimeric Antibodies

[0244] 1.1 Screening of Hybridomas for Anti-c-Kit Chimeric Antibodies

[0245] Four to six week old female Balb / C mice were selected and immunized with the D4D5 domain protein of human c-Kit antigen. After four immunizations, the mice were euthanized by cervical dislocation and the spleen and some peripheral lymph nodes were harvested. The spleen and peripheral lymph nodes were ground in DMEM medium to obtain a B cell rich suspension. An appropriate amount of the spleen and peripheral lymph node cell suspension was mixed with SP2 / 0 cells and subjected to electrofusion using a BTX electrocell manipulator. After electrofusion, the cells were cultured in ClonaCell HC medium (StemCell Technologies) and the supernatant was collected after 10 days and screened for the presence of antibodies against c-Kit by ELISA and FACS. TMHY Medium D (STEMCELL Technologies, 03805) was used for cell plating, IgG+ positive monoclonal was picked by Clonepix (Molecular Devices) to 96-well plate, after 2-3 days culture, the hybridoma supernatant was collected to screen the hybridoma clones which can bind to human and cynomolgus monkey c-Kit full-length antigen, and HEK293 cells stably expressing human and cynomolgus monkey c-Kit by ELISA and FACS.

[0246] TRNzol lysis method was used to extract the RNA of the candidate hybridoma monoclonal cells, and then single-stranded cDNA was synthesized by reverse transcription, and the variable region sequence of the antibody in the hybridoma monoclonal cells was amplified by using the above cDNA as a template. The light chain variable region of the above antibody was cloned into a vector containing human light chain constant region (pHCT2, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), and the heavy chain variable region was cloned into a vector containing human IgG4 heavy chain constant region (pHCT1s, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.). The plasmids were co-transfected into CHO-S cells. The complete IgG antibody was expressed and secreted into the culture medium. After 7 days of expression at 37°C, 5% CO2, 125 rpm, the supernatant was collected, and the chimeric IgG was purified by Protein A chromatography. The concentration of IgG was determined by spectrophotometry to obtain a human-mouse chimeric antibody for biological activity evaluation. The control antibody Barzolvolimab was synthesized according to the patent document CN106659782A, and was purified by Protein A chromatography after expression in CHO cells.

[0247] The amino acid sequence of the D4D5 domain protein of human c-Kit antigen is as follows:

[0248] 1.2 Screening of anti-c-Kit chimeric antibody by phage display library

[0249] The spleen and lymph nodes of mice were taken and total RNA was extracted after grinding and centrifugation. The mouse cells were lysed by adding 1 ml of Trizol lysis solution, and the supernatant was collected after centrifugation, then 200 ul of chloroform was added, vortexed vigorously for 15 s, and placed at room temperature for 3 min. After high-speed centrifugation, the upper aqueous phase was taken to a filter column, and an equal volume of 70% ethanol was added to the upper aqueous phase and mixed well. The upper aqueous phase was transferred to the binding column, washed with buffer, and then eluted with water to collect the RNA. After reverse transcription of the RNA, light and heavy chain specific primers were used for PCR amplification to obtain the light and heavy chain variable region cDNA library of the antibody, and the target fragments were recovered. The recovered fragments were double-digested with the vector, and the heavy and light chains were inserted into the vector using T4 DNA ligase, and then transformed into self-made DH5a competent cells. 3.5 g of each of the collected heavy / light chain library was used to extract plasmids, and the fragments were recovered by digestion and then ligated with the vector using T4 DNA ligase. The ligation product was purified and then electroporated into self-made TG1 competent cells, and the Fab phage library was obtained after infection with filamentous helper phage M13. Liquid phase panning was used, the phage was pre-incubated with biotin-labeled (thermo, 21338) c-Kit D4D5 for 1 h, and then added to the blocked streptavidin beads. After washing to remove non-specific binding or low affinity phage, the phage was eluted and infected TG1 bacteria, and then packaged again to obtain enriched phage for the next round of panning. The clones obtained after panning enrichment were inoculated into 96-well U-shaped well plates for culture and expression induction. The supernatant was taken for ELISA and FACS detection, and clones that could bind to human and cynomolgus monkey c-Kit D4D5 domain proteins were selected. Further FACS screening was used to select positive clones that could bind to HEK293 cells stably expressing human and cynomolgus monkey c-Kit.

[0250] After sequencing analysis of the positive clones, the light and heavy chain variable regions of the positive clones were cloned into a vector containing human heavy chain constant region and regulatory elements using DNA assembly method. After sequencing, the positive clones were transfected into CHO-S cells. The complete IgG antibody was expressed and secreted into the culture medium. After expression at 37°C, 5% CO2, 125 rpm for 7 days, the supernatant was collected, and the chimeric IgG was purified by Protein A chromatography. The concentration of IgG was determined by spectrophotometry to obtain the c-Kit chimeric antibody for biological activity evaluation.

[0251] The positive clones 67F1, 66D6, 118D8, 115I17 and 132J20 were obtained by the above screening, and the sequence information is as follows (wherein the CDR sequences are defined according to the Kabat definition rule).

[0252] 67F1

[0253] VH amino acid sequence:

[0254] Nucleotide sequence encoding VH:

[0255] HCDR1 : IFGIH (SEQ ID NO. 4)

[0256] HCDR2: VIWRGGSTDYNEAFMS (SEQ ID NO. 5)

[0257] HCDR3: YRYDRAY (SEQ ID NO. 6)

[0258] VL amino acid sequence:

[0259] Nucleotide sequence encoding VL:

[0260] LCDR1 : SASSSVSYMH (SEQ ID NO. 9)

[0261] LCDR2: STSNLAS (SEQ ID NO. 10)

[0262] LCDR3: QQRSSYPYT (SEQ ID NO. 11)

[0263] 66D6

[0264] VH amino acid sequence:

[0265] Nucleotide sequence encoding VH:

[0266] HCDR1 : NFGVQ (SEQ ID NO. 14)

[0267] HCDR2: VIWRGGSTDYNAAFMS (SEQ ID NO. 15) HCDR3: IRYSMDY (SEQ ID NO. 16)

[0268] VL amino acid sequence:

[0269] Nucleotide sequence encoding VL:

[0270] LCDR1 : SASSSVRYMY (SEQ ID NO. 19)

[0271] LCDR2: DTSNLVS (SEQ ID NO. 20)

[0272] LCDR3: QQRSSYPLT (SEQ ID NO. 21)

[0273] 118D8

[0274] VH Amino Acid Sequence:

[0275] Nucleotide Sequence Encoding VH:

[0276] HCDR1: NFGIH (SEQ ID NO. 24)

[0277] HCDR2: VIWRGGSTDYNAAFMS (SEQ ID NO. 15)

[0278] HCDR3: IQYAMDY (SEQ ID NO. 25)

[0279] VL Amino Acid Sequence:

[0280] Nucleotide Sequence Encoding VL:

[0281] LCDR1: SASSSVSYMH (SEQ ID NO. 9)

[0282] LCDR2: STSNLAS (SEQ ID NO. 10)

[0283] LCDR3: QQRNSYPYT (SEQ ID NO. 28)

[0284] 115I17

[0285] VH Amino Acid Sequence:

[0286] Nucleotide Sequence Encoding VH:

[0287] HCDR1: SFGIH (SEQ ID NO. 31)

[0288] HCDR2: VIWRGGSTDYNAAFMS (SEQ ID NO. 15)

[0289] HCDR3: IQYAMDY (SEQ ID NO. 25)

[0290] VL Amino Acid Sequence:

[0291] Nucleotide Sequence Encoding VL:

[0292] LCDR1:SASSSVSYMH(SEQ ID NO.9)

[0293] LCDR2: STSNLAS (SEQ ID NO.10)

[0294] LCDR3:QQRTSYPYT(SEQ ID NO.34)

[0295] 132J20

[0296] VH amino acid sequence:

[0297] Nucleotide sequence encoding VH:

[0298] HCDR1: NYYVH (SEQ ID NO.37)

[0299] HCDR2: WIFPGNVNTQYNESFKGKA(SEQ ID NO.38)

[0300] HCDR3:RGVNYAFDV(SEQ ID NO.39)

[0301] VL amino acid sequence:

[0302] Nucleotide sequence encoding VL:

[0303] LCDR1:TATSSVSSTFLH(SEQ ID NO.42)

[0304] LCDR2: STSNLAS (SEQ ID NO.10)

[0305] LCDR3:HQYHRSPYT(SEQ ID NO.43)

[0306] Example 2: Binding activity of anti-c-Kit chimeric antibody

[0307] 2.1 Binding activity of anti-c-Kit chimeric antibody to stable cell lines

[0308] The binding activity of candidate anti-c-Kit chimeric antibodies to human and cynomolgus monkey c-Kit HEK293 stable cell lines was detected using FACS. HEK293-human c-Kit and HEK293-cynomolgus monkey c-Kit cells in logarithmic growth phase were taken and their concentration adjusted to 5 x 10⁻⁶ cells with PBS containing 1% BSA. 5Cells / ml. Add 100 μl / well of cell suspension to a 96-well U-shaped plate, centrifuge at 300g for 5 min, discard the supernatant, add 100 μl of serially diluted chimeric antibody (starting concentration 200 nM, 5-fold dilution) to each well, and incubate at 4°C for 60 min. Centrifuge at 300g for 5 min, discard the supernatant, wash twice with PBS containing 1% BSA, add 50 μl / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) as secondary antibody, incubate on ice for 20 min, centrifuge at 300g for 5 min, discard the supernatant, wash twice with PBS containing 1% BSA, resuspend the cells in an appropriate amount of PBS, and detect the binding activity of the candidate chimeric antibody to the stable cell line using flow cytometry.

[0309] As shown in Figure 1, clones 66D6, 67F1, 118D8, 115I17, and 132J20 all exhibited strong binding ability.

[0310] 2.2 Binding activity of anti-c-Kit chimeric antibody to c-Kit recombinant antigen

[0311] Biomembrane interferometry (BLI / Gator prime) was used to detect the affinity of anti-c-Kit chimeric antibodies for human and cynomolgus monkey c-Kit recombinant antigens. Anti-c-Kit antibodies were captured using a Protein A probe, and then the human and cynomolgus monkey c-Kit recombinant antigens were used as analytes to detect the binding and dissociation signals with the probe. The results are shown in Table 1 below.

[0312] Table 1. Binding affinity of anti-c-Kit chimeric antibodies to human and cynomolgus monkey c-Kit recombinant antigens

[0313] The results showed that the chimeric antibody clones 66D6, 67F1, 118D8, 115I17 and 132J20 had stronger binding affinity to human and cynomolgus monkey c-Kit recombinant antigens than the control antibody Barzolvolimab.

[0314] Example 3. Inhibition of stem cell growth factor (SCF)-induced cellular phosphorylation by anti-c-Kit chimeric antibody.

[0315] After incubating HEK293 cells stably expressing human c-Kit with anti-c-Kit chimeric antibody at 37°C for 2 hours, gently aspirate the supernatant, add 500 ng / ml stem cell growth factor SCF (Peprotech, 300-07), stimulate at 37°C for 5 minutes, immediately centrifuge and discard the supernatant, add pre-chilled cell lysis buffer (containing 1 nM PMSF), lyse on ice for 5-10 minutes, centrifuge at 400g for 3 minutes, and transfer the supernatant from each well to the ELISA plate of the kit. Phospho-c-Kit (Tyr719) Sandwich ELISA Kit #7298L) according to the manufacturer's instructions.

[0316] The results are shown in Figure 2. Chimeric antibodies 115I17, 118D8 and 132J20 can effectively block SCF-induced c-Kit phosphorylation.

[0317] Example 4. Anti-c-Kit chimeric antibodies inhibit SCF-induced cell proliferation

[0318] M-07e cells belong to human megakaryocytic leukemia cell line and express human c-Kit. SCF can stimulate M-07e cell proliferation. In this example, the level of M-07e cell proliferation induced by c-Kit ligand SCF in the presence and absence of anti-c-Kit chimeric antibodies was evaluated. M-07e cells in logarithmic growth phase (Zhejiang Meisen Cells, CTCC-007-0239) were adjusted to a cell density of 2x10 5 cells / ml, 100 μl / well was added to a 96-well plate, and the cells were starved overnight. M-07e cells were incubated with anti-c-Kit chimeric antibodies before exposure to c-Kit ligand SCF, and after 2 h of incubation with gradient-diluted chimeric antibodies (starting concentration 200 nM, 4-fold dilution), 400 ng / ml SCF was added, and the cells were incubated at 37°C for 5 days. The number of M-07e cells was detected using Cell Titer reagent (Vazyme, DD1101-03) to indicate the level of cell proliferation.

[0319] The results are shown in Figure 3. Chimeric antibodies 66D6, 67F1, 118D8, 115I17 and 132J20 can effectively inhibit SCF-induced M-07e cell proliferation activity, and the inhibitory effect of chimeric antibodies 66D6 and 67F1 is significantly better than that of control antibody Barzolvolimab.

[0320] Example 5. Construction of anti-c-Kit humanized antibodies

[0321] The variable region sequences of anti-c-Kit chimeric antibodies 67F1 and 115I17 were humanized, CDR grafting was performed with the human germline gene with the highest homology, and then homology modeling was performed using a computer to analyze the CDR region and its surrounding framework amino acid sequences to avoid the concentration of molecular surface charge or hydrophobic regions. SPLEPA mutations (S228P, L235E and P329A) were introduced into Fc to completely remove the interaction with Fc gamma receptor and Clq. In addition, LA mutations (M428L and N434A) were introduced into the Fc of the antibody to prolong the half-life of the antibody. The sequences of humanized c-Kit antibodies h67F1 and h115I17 are shown below.

[0322] h67F1

[0323] VH amino acid sequence:

[0324] Nucleotide sequence encoding the VH:

[0325] HCDR1 : IFGIH (SEQ ID NO. 4)

[0326] HCDR2: VIWRGGSTDYNEALKS (SEQ ID NO. 46)

[0327] HCDR3: YRYDRAY (SEQ ID NO. 6)

[0328] Heavy chain amino acid sequence:

[0329] Nucleotide sequence encoding the heavy chain:

[0330] VL amino acid sequence:

[0331] Nucleotide sequence encoding the VL:

[0332] LCDR1 : RASSSVSYLH (SEQ ID NO. 51 )

[0333] LCDR2: STSNLAS (SEQ ID NO. 10)

[0334] LCDR3: QQRSSYPYT (SEQ ID NO. 11 )

[0335] Light chain amino acid sequence:

[0336] Nucleotide sequence encoding the light chain:

[0337] h115I17

[0338] VH amino acid sequence:

[0339] Nucleotide sequence encoding the VH:

[0340] HCDR1 : SFGIH (SEQ ID NO. 31 )

[0341] HCDR2: VIWRGGSTDYNAALKS (SEQ ID NO. 56)

[0342] HCDR3: IQYAMDY (SEQ ID NO. 25)

[0343] Heavy chain amino acid sequence:

[0344] Nucleotide sequence encoding the heavy chain:

[0345] VL amino acid sequence:

[0346] Nucleotide sequence encoding the VL:

[0347] LCDR1: RASSSVSYMH (SEQ ID NO. 61)

[0348] LCDR2: STSNLAS (SEQ ID NO. 10)

[0349] LCDR3: QQRTSYPYT (SEQ ID NO. 34)

[0350] Light chain amino acid sequence:

[0351] Nucleotide sequence encoding the light chain:

[0352] Example 6. Binding activity of anti-c-Kit humanized antibodies

[0353] 6.1 Binding activity of anti-c-Kit humanized antibodies to stable cell lines

[0354] The binding activity of anti-c-Kit humanized antibodies to human and cynomolgus monkey c-Kit HEK293 stable cell lines was detected by FACS. Logarithmic growth phase of HEK293-human c-Kit and HEK293-cynomolgus monkey c-Kit cells were taken, respectively, and the cell concentration was adjusted to 5x10 5Cells were plated at 1 x 105cells / ml, 100 μl / well into 96-well U-bottom plates, 300 g centrifugation for 5 min, and the supernatant was discarded. Each well was added with 100 μl of gradient dilution of chimeric antibody (initial concentration 200 nM, 5-fold dilution), and incubated at 4°C for 60 min. 300 g centrifugation for 5 min, and the supernatant was discarded. The cells were washed twice with PBS containing 1% BSA. Secondary antibody was added with 50 μl / well of Alexa Fluro 647 labeled goat anti-human IgG Fc (1:300 dilution), and incubated at 4°C for 20 min. 300 g centrifugation for 5 min, and the supernatant was discarded. The cells were washed twice with PBS containing 1% BSA. After resuspension with appropriate amount of PBS, the binding activity of the candidate humanized antibody to the stable cell line was detected by flow cytometry.

[0355] The results are shown in Figure 4. Both humanized antibodies h67F1 and h115I17 can bind to human and cynomolgus monkey c-Kit.

[0356] 6.2 Binding activity of anti-c-Kit humanized antibodies to c-Kit recombinant antigen

[0357] The affinity of anti-c-Kit humanized antibodies to human and cynomolgus monkey c-Kit recombinant antigen was detected by biofilm interference technology (BLI / Gator prime). Anti-c-Kit humanized antibodies were captured by Protein A probe, and then human and cynomolgus monkey c-Kit recombinant antigen was used as analyte to detect the binding and dissociation signals of the probe. The results are shown in Table 2 below.

[0358] Table 2. Binding affinity of anti-c-Kit humanized antibodies to human and cynomolgus monkey c-Kit recombinant antigen

[0359] The results show that both humanized antibodies h67F1 and h115I17 can bind to human and cynomolgus monkey c-Kit recombinant antigen, and the affinity is stronger than that of the control antibody Barzolvolimab.

[0360] Example 7 Anti-c-Kit humanized antibodies inhibit SCF-induced cell proliferation

[0361] Logarithmic growth phase M-07e cells were taken, and the cell density was adjusted to 2e 5 cells / ml, 100 μl / well was added to a 96-well plate, and the cells were starved overnight. M-07e cells were incubated with anti-c-Kit antibodies before exposure to c-Kit ligand SCF. Gradient dilution of humanized antibodies (initial concentration 200 nM, 4-fold dilution) was added and incubated for 2 h, and then 400 ng / ml SCF was added. The cells were incubated at 37°C for 5 days. The number of M-07e cells was detected by Cell Titer reagent to indicate the level of proliferation.

[0362] The results are shown in Figure 5. Both humanized antibodies h67F1 and h115I17 were able to inhibit SCF-induced M-07e cell proliferation, and the inhibitory activity of h67F1 antibody was stronger than that of the control antibody Barzolvolimab.

[0363] Example 8 Anti-c-Kit humanized antibodies inhibit mast cell degranulation activity

[0364] Mast cells were obtained by in vitro induction culture of human PBMC-derived CD34-positive cells. The induced mast cells were taken, the cell density was adjusted to 2x10 5 The supernatant was transferred to a clean 96-well plate, and the absorbance of the supernatant was detected after subsequent reaction with a substrate.

[0365] After the supernatant was transferred, the cells in each well were washed once with 200 μl of Tyrode's solution, 100 μl / well of 3% Triton lysis solution was added, and lysis was performed on ice for 5-10 min. After lysis, centrifugation was performed at 400 g for 3 min, and 70 μl / well of the lysis solution was transferred to a clean 96-well plate for subsequent reaction with a substrate and detection of the absorbance of the lysis solution.

[0366] The method for detecting the absorbance is as follows: 35 μl / well of substrate solution (4-nitrophenyl 2-(acetylamino)-2-deoxy-β-D-glucopyranoside, Mcllvaine, N8146, 90-500 mg) was added to the supernatant and lysis solution, respectively, and the reaction was performed at 37°C for 2 h. The degranulation result was detected using an enzyme label instrument, and the reading wavelength was set to 405 nm.

[0367] The release rate of β-hexosaminidase was calculated according to the following formula:

[0368] The release rate of β-hexosaminidase (%) = supernatant absorbance / (supernatant absorbance + lysis solution absorbance) * 100

[0369] The inhibitory activity of the anti-c-Kit humanized antibodies on mast cell degranulation was determined according to the size of the β-hexosaminidase release rate. The results are shown in Figure 6 and Table 3.

[0370] Table 3. β-hexosaminidase release rate of anti-c-Kit humanized antibodies

[0371] The anti-c-Kit humanized antibodies h67F1 and h115I17 can inhibit mast cell degranulation activity at each dose, and the inhibition of mast cell degranulation activity is better than that of the control antibody Barzolvolimab at a high dose.

[0372] Example 9 Anti-c-Kit humanized antibodies inhibit SCF-induced cytokine release

[0373] Mast cells were obtained by in vitro induction culture of human PBMC-derived CD34-positive cells. The induced mast cells were taken, the cell density was adjusted to 2x10 5 cells / ml, 100 μl / well was added to a 96-well flat-bottom plate, and the cells were cultured overnight using blank medium to deplete the cytokines in the cell induction system. The next day, the medium was replaced with a medium containing only SCF for culture. At the same time, blank wells were set up and cultured using blank medium. 50 μl of humanized antibody samples of different concentrations (10 nM and 0.1 nM) were added to each well, and after 48 h of culture at 37°C, the supernatant was detected using a TNFα detection kit (BD Bioscience, 1266709) to determine the degree of cytokine release. The results are shown in Figure 7 and Table 4.

[0374] Table 4. Anti-c-Kit humanized antibodies inhibit SCF-induced TNFα release

[0375] The results show that the anti-c-Kit humanized antibodies h67F1 and h115I17 can effectively inhibit the SCF-induced cytokine release activity of mast cells at each concentration, and the inhibitory activity is better than that of the control antibody Barzolvolimab.

[0376] Example 10 Pharmacokinetics of anti-c-Kit humanized antibodies in FcRn humanized mice

[0377] Five FcRn humanized mice (Biosail Biopharma Co., Ltd.; Cat No.: 110001) were each given 10 mg / kg of anti-c-Kit humanized antibody by subcutaneous injection. On days 1, 2, 3, 5, 7, 14, 21, 28, 35, 42, 49, 56 after administration, about 100 μl of blood samples were collected from each mouse. The concentration of anti-c-Kit humanized antibody in serum was measured by ELISA. The human c-Kit antigen was coated on the enzyme-labeled plate as a capture reagent, and after blocking, the diluted standard curve sample and the sample to be tested were added to the enzyme-labeled plate for incubation. After washing away the free anti-c-Kit humanized antibody, secondary antibody (Mouse Anti-Human IgG4 Fc-HRP) was added for incubation, and after washing away the excess secondary antibody, substrate was added for color development, and stop solution was added to terminate the color development reaction. The OD value was read at a wavelength of 450 nm (reference wavelength of 650 nm). The pharmacokinetic parameters were calculated using the non-compartment model of Winnonlin software.

[0378] The results are shown in Figure 8, and the half-life of anti-c-Kit humanized antibody h67F1 in FcRn humanized mice was 12.1 ± 0.6 days.

[0379] Example 11 Effect of anti-c-Kit humanized antibody in a mouse systemic passive sensitization model

[0380] A systemic passive sensitization model was used to simulate urticaria in mice by activating mast cells through cross-linking IgE, leading to the release of related inflammatory factors and proteases. HuHSC-NCG-SGM3 mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd.; Strain NO.: T056601) of 14-18 weeks of age were selected, and each mouse was sensitized by tail vein injection of Anti-DNP IgE (Sigma, D8406) at a dose of 1.6 ug. After 24 hours, each mouse was challenged by tail vein injection of DNP-HAS (Sigma, A6661) at a dose of 500 ug. The anal temperature was detected before DNP-HAS challenge, and every 10 minutes after DNP-HAS challenge for a total of 1 hour. The behavior of the mice was recorded every 10 minutes after DNP-HAS challenge for a total of 1 hour, and a clinical score was given. The scoring criteria were as follows: 1 point: scratching 2 points: piloerection, facial edema 3 points: dyspnea 4 points: coma, unresponsive 5 points: death. Antibodies h67F1, Barzolvolimab, or isotype control were administered by subcutaneous injection 24 hours before tail vein injection of Anti-DNP IgE for sensitization.

[0381] Results of administering the antibodies at 10 mg / kg and 3 mg / kg are shown in Figures 9 and 10. n = 4 for the isotype control group and n = 6 for the other groups. Figure 11 shows results of administering the antibodies at 10 mg / kg and 30 mg / kg, where n = 2 for the isotype control group; n = 5 for the h67F1 30 mg / kg and Barzolvolimab 30 mg / kg groups; and n = 3 for the h67F1 10 mg / kg and Barzolvolimab 10 mg / kg groups.

[0382] Results are shown in Figure 9. After DNP-HAS challenge, the body temperature of the mice in each group began to decrease. More than half of the mice in the isotype control group died at 30 min (indicated by symbols in the figure), and the body temperature of the mice in this group was no longer recorded. In each experimental group, the h67F1 10 mg / kg group significantly inhibited the decrease in body temperature, and the inhibitory effect was better than that of the Barzolvolimab 10 mg / kg group. The h67F1 3 mg / kg group inhibited the decrease in body temperature better than the Barzolvolimab 3 mg / kg group. In terms of clinical scores, the results are shown in Figure 10. The mice in the isotype control group had high clinical scores, and all of the mice died at 60 min. The scores of the h67F1 3 mg / kg and 10 mg / kg groups were lower than those of the Barzolvolimab groups at the same dose. In addition, as shown in Figure 11, more than half of the mice in the isotype control group died at 30 min at a dose of 30 mg / kg. At doses of 30 mg / kg and 10 mg / kg, the h67F1 inhibited the decrease in body temperature better than the Barzolvolimab groups at the corresponding doses, and the 30 mg / kg group of h67F1 had significantly better inhibitory activity than the 10 mg / kg group.

[0383] The above results show that the anti-c-Kit humanized antibody h67F1 can dose-dependently inhibit passive systemic anaphylaxis in mice, and the inhibitory activity is better than that of the control antibody Barzolvolimab.

[0384] While the preferred embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that many changes, modifications, and substitutions can be made thereto without departing from the application. It is to be understood that various alternatives to the embodiments described herein can be employed in practicing the present application. The following claims are intended to cover all such alternatives and equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that binds c-Kit, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 4, 46, 6, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 51, 10, 11, respectively; or (2) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 31, 56, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 61, 10, 34, respectively; or (3) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, 6, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 9, 10, 11, respectively; or (4) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, 16, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 19, 20, 21, respectively; or (5) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 24, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 9, 10, 28, respectively; or (6) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 31, 15, 25, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 9, 10, 34, respectively; or (7) the VH comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 37, 38, 39, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 42, 10, 43, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein: (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:44, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:49; or (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:59; or (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7; or (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17; or (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26; or (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:32; or (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 35, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

40.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 44, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 49; or (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 54, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 59; or (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 2, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 7; or (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 12, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 17; or (5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 22, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 26; or (6) the VH comprises the amino acid sequence set forth in SEQ ID NO: 29, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 32; or (7) the VH comprises the amino acid sequence set forth in SEQ ID NO: 35, and the VL comprises the amino acid sequence set forth in SEQ ID NO:

40.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody comprises: (i) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 47, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 52; or (ii) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

62.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody comprises: (i) a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 47, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 52; or (ii) a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 57, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO:

62.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the antibody is a bispecific antibody further comprising a second antigen-binding region that binds to a second antigen.

12. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-11.

13. The nucleic acid according to claim 12, comprising: (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50; or (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60; or (3) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8; or (4) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 13, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18; or (5) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27; or (6) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 30, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33; or (7) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

41.

14. The nucleic acid according to claim 12, comprising (1) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 48, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 53; or (2) a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 58, and a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:

63.

15. A vector comprising the nucleic acid according to any one of claims 12-14.

16. A host cell comprising the nucleic acid according to any one of claims 12-14 or the vector according to claim 15.

17. A pharmaceutical composition comprising (i) the antibody or antigen binding fragment thereof according to any one of claims 1-11; and (ii) a pharmaceutically acceptable carrier or excipient.

18. The pharmaceutical composition according to claim 17, further comprising a second therapeutic agent.

19. The pharmaceutical composition according to claim 18, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

20. A conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11, and a chemical moiety conjugated thereto.

21. A chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11.

22. A method for treating a disease in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-11, a pharmaceutical composition according to any one of claims 17-19, a conjugate according to claim 20, or a CAR according to claim 21.

23. The method of claim 22, wherein the disease is a disease associated with c-Kit expression, such as an allergic disease, gastrointestinal stromal tumor, systemic mastocytosis, eosinophilic esophagitis, sickle cell disease, hematopoietic system disease, severe combined immunodeficiency, myelodysplastic syndrome, chronic granulomatous disease, diabetic macular edema, wet age-related macular degeneration.

24. The method of claim 23, wherein the allergic disease is selected from the group consisting of urticaria, nodular prurigo, asthma.

25. The method of claim 24, wherein the urticaria is chronic urticaria, such as chronic spontaneous urticaria or chronic inducible urticaria.

26. The method according to any one of claims 22-25, further comprising administering to the subject a second therapeutic agent.

27. The method according to claim 26, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

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