Anti-kit antibody and pharmaceutical use thereof

Abstract

The present invention relates to an anti-KIT antibody and pharmaceutical use thereof. Specifically, the present invention relates to an anti-KIT antibody and use thereof in preparing a medicament for preventing or treating a disease such as an inflammatory disease and autoimmune disease.

Description

Anti-kit antibodies and medical uses thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to anti-KIT antibodies and medical uses thereof. BACKGROUND

[0002] The statements herein are provided only to aid in the understanding of the present disclosure, and do not necessarily constitute prior art.

[0003] KIT, also known as CD117, can specifically bind stem cell factor SCF. The structure of KIT includes an extracellular region, a transmembrane region and an intracellular region. The extracellular region consists of 5 immunoglobulin-like domains, the first 3 domains are involved in the binding of SCF, and the 4th and 5th domains are involved in receptor dimerization. The intracellular region has receptor tyrosine kinase activity. Under normal conditions, KIT exists in monomer form, dimerizes and autophosphorylates after binding to the ligand SCF, thereby activating downstream signaling pathways and participating in cell processes such as cell proliferation, differentiation, apoptosis and migration.

[0004] Mast cells are widely distributed around the microvessels of the skin and the submucosa of the internal organs, can secrete a variety of cytokines and release allergic mediators, and have immunoregulatory effects. Mast cells are a unique tissue-resident immune cell and an important effector cell that causes many allergic and autoimmune diseases. KIT is one of the important receptors on the surface of mast cells, and KIT activation is closely related to the growth, survival, differentiation and maturation, and homing of mast cells. SCF binding to KIT receptor can enhance mast cell degranulation and cytokine release caused by antigens. Therefore, KIT related to mast cell activation can be used as a drug target for the treatment of certain allergic or autoimmune diseases, such as urticaria.

[0005] The existing patents WO2007127317A1, WO2014018625A1, WO2021107566A1, WO2022159737A1, WO2020112687, etc. disclose anti-KIT antibodies. SUMMARY

[0006] The present disclosure provides an anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0007] I, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 26, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 27; or

[0008] II, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 15; or

[0009] III, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 32, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 33; or

[0010] IV, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 16, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 17.

[0011] The present disclosure also provides an anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0012] V, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 38, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 39, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; wherein

[0013] SEQ ID NO: 38 is LCDR1 as set forth in X1ASSSVSYMH, XI is R or S;

[0014] SEQ ID NO: 39 is LCDR2 as set forth in STSNLAX2, X2 is D or S;

[0015] or

[0016] VI, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0017] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, comprising a heavy chain variable region and a light chain variable region, wherein:

[0018] I, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 36, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 37, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; or

[0019] II, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 5, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; or

[0020] III, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0021] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the anti-KIT antibody is a murine, chimeric, humanized, or fully human antibody.

[0022] In some embodiments, the anti-KIT antibody of any of the preceding claims, wherein the anti-KIT antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0023] In some embodiments, the anti-KIT antibody of any of the preceding claims, wherein the anti-KIT antibody is a humanized antibody.

[0024] In some embodiments, the anti-KIT antibody of any of the preceding claims, comprising a heavy chain variable region and a light chain variable region, wherein:

[0025] I. the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27 or an amino acid sequence having at least 75% sequence identity thereto; or

[0026] II. the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence having at least 75% sequence identity thereto; or

[0027] III. the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence having at least 75% sequence identity thereto; or

[0028] IV. the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence having at least 75% sequence identity thereto.

[0029] The above having at least 75% sequence identity to an antibody heavy chain variable region or light chain variable region includes, but is not limited to, having at least 75%, 77%, 80%, 82%, 84%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity in the framework region of the antibody.

[0030] In some embodiments, the anti-KIT antibody of any of the preceding claims, comprising a heavy chain variable region and a light chain variable region, wherein:

[0031] I, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical thereto; or

[0032] II, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical thereto; or

[0033] III, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence that is at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence that is at least 90% identical thereto; or

[0034] IV, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical thereto.

[0035] The above having at least 90% sequence identity to an antibody heavy chain variable region or light chain variable region includes, but is not limited to, having, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity in the framework region of the antibody.

[0036] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the anti-KIT antibody is an antibody fragment.

[0037] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody fragment of the anti-KIT antibody is selected from a Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0038] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region and a light chain constant region.

[0039] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region derived from a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region and a light chain constant region that is a human kappa or lambda light chain constant region.

[0040] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region derived from a human IgGl heavy chain constant region and a light chain constant region that is a human kappa or lambda light chain constant region.

[0041] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region derived from a human IgGl heavy chain constant region and a light chain constant region that is a human kappa light chain constant region; the human IgGl heavy chain constant region comprises mutations at positions 234, 235, 237, 428 and 434 (according to the EU numbering convention, hereinafter).

[0042] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region derived from a human IgGl heavy chain constant region and a light chain constant region that is a human kappa light chain constant region; the human IgGl heavy chain constant region comprises mutations L234A, L235A, G237A, M428L and N434S (according to the EU numbering convention, hereinafter).

[0043] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region comprising an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain constant region comprising an amino acid sequence as set forth in SEQ ID NO: 19.

[0044] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the anti-KIT antibody comprises a heavy chain and a light chain, wherein:

[0045] I, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 28 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 29 or an amino acid sequence having at least 85% sequence identity thereto; or

[0046] II, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence having at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 21 or an amino acid sequence having at least 85% sequence identity thereto; or

[0047] III, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence that has at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85% sequence identity thereto; or

[0048] IV, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 22 or an amino acid sequence that has at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 23 or an amino acid sequence that has at least 85% sequence identity thereto.

[0049] The above having at least 85% sequence identity to an antibody heavy chain or light chain includes, but is not limited to, having, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0050] In another aspect, the present disclosure also provides a multispecific antibody comprising the anti-KIT antibody of any of the preceding aspects.

[0051] In another aspect, the present disclosure provides an anti-KIT antibody that competes for binding to human KIT with the anti-KIT antibody of any of the above aspects or the multispecific antibody described above.

[0052] In another aspect, the present disclosure provides an anti-KIT antibody that binds to the same KIT antigenic epitope as the anti-KIT antibody of any of the above aspects or the multispecific antibody described above.

[0053] In another aspect, the present disclosure provides a pharmaceutical composition comprising the anti-KIT antibody of any of the above aspects or the multispecific antibody described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0054] In another aspect, the present disclosure provides an isolated nucleic acid encoding the anti-KIT antibody of any of the above aspects or the multispecific antibody described above.

[0055] In another aspect, the present disclosure provides a vector comprising the isolated nucleic acid described above.

[0056] In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid described above.

[0057] In another aspect, the present disclosure provides a method for producing the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, comprising culturing the host cell of the above in a culture medium to form and accumulate the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, and the step of recovering the antibody from the culture.

[0058] In another aspect, the present disclosure provides the use of the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, or the pharmaceutical composition of the above, in the manufacture of a medicament for preventing or treating a disease or disorder associated with KIT.

[0059] In another aspect, the present disclosure provides a method of preventing or treating a disease or disorder associated with KIT, comprising administering to a subject the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, or the pharmaceutical composition of the above.

[0060] In another aspect, the present disclosure provides the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, or the pharmaceutical composition of the above, for use as a medicament.

[0061] In some embodiments, the disease or disorder associated with KIT of any one of the above is selected from the group consisting of an inflammatory disease, an ocular disease, a cancer, a hematological disease, an allergy, and an autoimmune disease.

[0062] In some embodiments, the disease or disorder associated with KIT of any one of the above is selected from the group consisting of prurigo nodularis, esophagitis, asthma, atopic dermatitis, urticaria, myeloma, connective tissue tumor, leukemia, lung cancer, gastrointestinal stromal tumor, hemoglobinopathy, retinal disorder, diabetic macular edema, and wet age-related macular degeneration.

[0063] In some embodiments, the disease or disorder associated with KIT of any one of the above is urticaria.

[0064] In some embodiments, the disease or disorder associated with KIT of any one of the above is chronic urticaria; preferably, the chronic urticaria is selected from the group consisting of chronic spontaneous urticaria, chronic inducible urticaria, and cold contact urticaria.

[0065] In another aspect, the present disclosure provides a method of preventing or treating a disease or disorder, comprising administering to a subject the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, or the pharmaceutical composition of the above.

[0066] In another aspect, the present disclosure provides the use of the anti-KIT antibody of any one of the above, or the multispecific antibody of the above, or the pharmaceutical composition of the above, in the manufacture of a medicament for preventing or treating a disease or disorder.

[0067] In another aspect, the present disclosure provides use of an anti-KIT antibody of any one of the above, or a multispecific antibody of the above, or a pharmaceutical composition of the above, in the manufacture of a medicament for preventing or treating a disease or condition selected from the group consisting of an inflammatory disease, an ocular disease, a cancer, a hematological disease, an allergy, and an autoimmune disease.

[0068] In another aspect, the present disclosure provides use of an anti-KIT antibody of any one of the above, or a multispecific antibody of the above, or a pharmaceutical composition of the above, in the manufacture of a medicament for preventing or treating a disease or condition selected from the group consisting of an inflammatory disease, an ocular disease, a cancer, a hematological disease, an allergy, and an autoimmune disease.

[0069] In another aspect, the present disclosure provides use of an anti-KIT antibody of any one of the above, or a multispecific antibody of the above, or a pharmaceutical composition of the above, in the manufacture of a medicament for preventing or treating a disease or condition selected from the group consisting of an inflammatory disease, an ocular disease, a cancer, a hematological disease, an allergy, and an autoimmune disease.

[0070] In some embodiments, the urticaria is chronic urticaria; preferably, the chronic urticaria is selected from the group consisting of chronic spontaneous urticaria, chronic inducible urticaria, and cold contact urticaria.

[0071] In some embodiments, the inflammatory disease is selected from the group consisting of nodular prurigo, esophagitis, asthma, atopic dermatitis, and chronic urticaria; the cancer is selected from the group consisting of myeloma, connective tissue tumor, leukemia, lung cancer, and gastrointestinal stromal tumor; the hematological disease is hemoglobinopathy.

[0072] In some embodiments, the hemoglobinopathy is selected from the group consisting of sickle cell disease and beta-thalassemia; the chronic urticaria is selected from the group consisting of chronic spontaneous urticaria, chronic inducible urticaria, and cold contact urticaria; the lung cancer is small cell lung cancer; the leukemia is chronic myelocytic leukemia or acute myeloid leukemia; the esophagitis is eosinophilic esophagitis.

[0073] In some embodiments, the disease or condition of any one of the above is selected from the group consisting of retinal disorder, diabetic macular edema, wet age-related macular degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1A: Comparison of the activity of humanized antibody 531-Hu and control antibody CDX-0159 in inhibiting mast cell secretion of GM-CSF.

[0075] Figure 1B: Comparison of the activity of humanized antibody 4A11-Hu and control antibody CDX-0159 in inhibiting mast cell secretion of GM-CSF.

[0076] Figure 2: Pharmacodynamic comparison of humanized antibody 531-Hu and control antibody CDX-0159 degranulation of canine skin mast cells. DETAILED DESCRIPTION

[0077] TERMINOLOGY

[0078] For the purposes of the present disclosure, certain technical and scientific terms are described below. Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0079] As used in the specification and claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0080] Unless the context clearly indicates otherwise, throughout the patent specification and claims, the words "comprise", "have" and "include" and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense.

[0081] The term "and / or" means both "and" and "or". For example, the phrase "A, B, and / or C" is intended to cover the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0082] The three letter code and one letter code for amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p 3558 (1968).

[0083] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine, and modified forms of those amino acids. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bonded to a hydrogen, a carboxyl, an amino, and an R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise function in a manner similar to naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0084] The term "amino acid mutation" includes amino acid substitutions (also referred to as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as reduced or no binding to an Fc receptor. Amino acid sequence deletions and insertions include deletions and insertions at the amino- and / or carboxy-terminus of a polypeptide chain. A particular amino acid mutation can be an amino acid substitution. In some embodiments, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5- hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods other than genetic engineering to alter the side chain groups of amino acids, such as chemical modifications, are also contemplated to be useful. Various names can be used herein to refer to the same amino acid mutation. In this regard, a particular amino acid residue at a position can be denoted in the position + amino acid residue format, e.g., 428L, to indicate that the amino acid residue at position 428 is L. M428L indicates that the amino acid residue at position 428 has been mutated from M to L. It is understood that when an amino acid sequence is defined in a claim in the position + residue format, the amino acid prior to mutation at that position does not limit the claim. In this regard, "an Fc region comprising amino acid mutations 428L and 434S" indicates that the Fc region has amino acid mutations at position 428 to lysine (L) and at position 434 to serine (S).

[0085] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0086] The term "antibody" is used in the broadest sense, and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antibody fragments (or antigen binding fragments, or antigen binding portions), so long as they exhibit the desired antigen-binding activity.

[0087] The term "antigen binding fragment" encompasses full length antibodies, Fabs, modified Fabs, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single domain antibodies (sdAbs, e.g., VH or VL or VHH), single chain Fabs (scFabs), single chain antibodies (e.g., scFv, sc(Fv)2), diabodies, linear antibodies, bi- or tri- or tetra-valent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope binding fragments of any of the above. Methods for generating and making these antigen binding fragments are well known in the art.

[0088] A "native antibody" refers to an immunoglobulin molecule naturally occurring. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain, followed by a constant light (CL) domain.

[0089] The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain with an Fc region as defined herein. A native intact antibody light chain includes a light chain variable region, VL, at the amino-terminal end of the light chain, and a constant region, CL, including kappa and lambda chains; a heavy chain includes a variable region, VH, at the amino-terminal end of the heavy chain, and a constant region at the carboxy-terminal end, where CH3 is closest to the carboxy-terminal end of the polypeptide, and the heavy chain can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0090] The term "Fc region" or "fragment crystallizable region" is used to define a C-terminal region of an antibody heavy chain, including native and engineered Fc regions. In some embodiments, the Fc region comprises two subunits, which are identical or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as stretching from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. Suitable Fc regions for use in the antibodies described herein include Fc regions of human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also be varied, for example by deleting the C-terminal lysine (residue 447 according to EU numbering system) or both C-terminal glycine and lysine (residues 446 and 447 according to EU numbering system) of an Fc region. Unless otherwise specified, numbering of the Fc region is EU numbering system, also known as EU index.

[0091] The Fc region can be suitably obtained by partially digesting an IgG monoclonal antibody or the like with a proteolytic enzyme such as pepsin, followed by eluting the fraction adsorbed to a protein A or protein G column. As the proteolytic enzyme, an enzyme that can digest a full-length antibody to produce Fab, F(ab')2 in a limited manner by suitably setting the reaction conditions of the enzyme such as pH is used, and there is no particular limitation, and for example, pepsin, papain, or the like can be exemplified.

[0092] The term "variable region" or "variable domain" of an antibody refers to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. In this document, the variable regions of an antibody heavy chain (VH) and light chain (VL) each comprise four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Within the variable region, the term "complementarity determining region" or "CDR" refers to a region that is primarily responsible for mediating specific antigen binding; "framework" or "FR" refers to the variable domain residues other than those in the CDRs. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; and the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each of the VH and VL is composed of three CDRs and four FRs, arranged from an amino-terminus (also referred to as N-terminus) to a carboxy-terminus (also referred to as C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0093] The boundaries of the CDRs can be determined by various known schemes, such as the "Kabat" numbering convention, the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention, and the like; the correspondence between the various numbering systems is well known to those skilled in the art.

[0094] Unless otherwise specified, the variable regions and CDRs in the embodiments of the present disclosure are in accordance with the "Kabat" numbering convention.

[0095] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibody, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0096] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different, distinct source or species.

[0097] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having less immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart (i.e., the constant region as well as the framework region portion of the variable region).

[0098] The terms "human antibody," "humanized antibody," "fully human antibody," "fully human antibody" are used interchangeably and mean an antibody in which the variable and constant regions are of human sequence. The term encompasses antibodies derived from human genes but which have had sequences altered, e.g., to reduce possible immunogenicity, increase affinity, eliminate a cysteine or glycosylation site that can cause undesirable folding, etc. The term encompasses these antibodies that are recombinantly produced in non-human cells, which can impart glycosylation not characteristic of human cells. The term also encompasses antibodies that have been produced in transgenic mice that contain some or all human immunoglobulin heavy and light chain loci. The meaning of human antibody expressly excludes humanized antibodies.

[0099] The term "affinity" refers to the overall strength of the noncovalent interactions between individual binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, binding "affinity" refers to intrinsic binding affinity, which reflects the 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its ligand Y can generally be represented by the dissociation constant (KD). Affinity can be measured by routine methods known in the art, including those described herein.

[0100] The term "kassoc" or "ka" as used herein refers to the association rate of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). KDvalues for antibodies can be determined using methods well known in the art. For example, affinity can be measured in solution using surface plasmon resonance (e.g., Biacore), using a Biacore® biosensor system, e.g., a Biacore 2000® or a Biacore 3000® system, or by solution equilibrium titration (SET).

[0101] The term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time interactions between biological molecules. Changes in protein concentrations within a biosensor matrix are detected by detecting local refractive index changes using the BIAcore system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0102] The term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence mutated Fc region) and which vary with the antibody isotype. Examples of antibody effector functions include but are not limited to: Clq binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0103] The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that can be present in minor amounts. In contrast, polyclonal antibodies typically include a population of different antibodies that are typically specific for different epitopes. "Monoclonal" should not be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

[0104] The term "bispecific antibody" refers to an antibody (including an antibody or antigen-binding fragment thereof, such as a single chain antibody) that is capable of specifically binding to two different antigens or at least two different epitopes of the same antigen. Various structures of bispecific antibodies have been disclosed in the prior art, which can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies according to the integrity of IgG molecules, into bivalent, trivalent, tetravalent or more valent bispecific antibodies according to the number of antigen-binding regions, and into symmetric structure bispecific antibodies and asymmetric structure bispecific antibodies according to whether the structure is symmetric. Among them, the bispecific antibody based on antibody fragments, such as Fab fragments lacking Fc fragments, forms a bispecific antibody by combining 2 or more Fab fragments in one molecule, which has lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration; IgG-like bispecific antibodies (e.g., with Fc fragments), which have relatively large molecular weight, the Fc fragment helps to purify the antibody and improves its solubility, stability, and the Fc part can also bind to the receptor FcRn to increase the serum half-life of the antibody.

[0105] The term "antigen" refers to a molecule or a portion of a molecule that is capable of being selectively bound by, for example, an antigen-binding protein (including, e.g., an antibody). The antigen can have one or more epitopes that are capable of interacting with different antigen-binding proteins (e.g., antibodies).

[0106] The term "epitope" refers to a region (area or region) on an antigen that is capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope can be formed by contiguous amino acids (linear epitopes) or comprise non-contiguous amino acids (conformational epitopes) that come into spatial proximity as a result of the folding of the antigen. The difference between a conformational epitope and a linear epitope is that the binding of an antibody to a conformational epitope is lost in the presence of a denaturing solvent. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide mapping, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens, and cross-blocking.

[0107] The term "capable of specifically binding," "specifically binding," or "binding" refers to the ability of an antibody to bind to a certain antigen or epitope thereof with higher affinity than to other antigens or epitopes. Typically, an antibody binds to a certain antigen or epitope thereof with a dissociation constant (Kd) of about 1 x 10 -6 M or less (e.g., about 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9antibody binds to an antigen or an epitope thereof with a dissociation equilibrium constant (KD) of 10"7M or less. In some embodiments, the antibody binds to an antigen with a KD that is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by An antibody that specifically binds to an antigen or an epitope thereof does not exclude cross-reactivity to other related antigens, for example, cross-reactivity to a corresponding antigen from another species (homologous), such as a human or a monkey, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0108] The term "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells (NK), monocytes, macrophages, and neutrophils, via Fcy receptors (FcyRs) expressed on the effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcyRIIIa. The ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using cells expressing an antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a native intracellular protein) from the lysed cells.

[0109] The term "antibody-dependent cellular phagocytosis" (ADCP) refers to a mechanism of eliminating antibody-coated target cells through internalization by phagocytic cells, such as macrophages or dendritic cells.

[0110] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates complement component Clq, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of the target cell, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0111] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties to the reference nucleotide, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0112] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid encoding a polypeptide is one that is

[0113] The term "identity" with respect to a sequence refers to the extent to which the amino acid / nucleic acid of two sequences are the same at equivalent positions when the two sequences are optimally aligned; if necessary, gaps are introduced to achieve maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. To determine percent sequence identity, alignments can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One of skill in the art can determine appropriate parameters to be used in measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0114] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, e.g., a adenoviral associated virus vector (AAV or AAV2), wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. The term "expression vector" or "expression construct" refers to a vector that can be transformed into a host cell, and which contains nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions to which they are operably linked, along with the host cell. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, where introns are present, RNA splicing of coding regions operably linked thereto.

[0115] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The progeny can not be completely identical to the parent cell from which it was derived, but may, for example, contain mutations. Mutant progeny that have the same function or biological activity as the parent cell are included herein. Host cells include prokaryotic and eukaryotic host cells, including but not limited to mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus sp., Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa, and Yarrowia lipolytica.

[0116] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0117] The term "pharmaceutical composition" denotes a mixture of one or more of the antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0118] The term "pharmaceutically acceptable carrier, diluent or excipient" refers to a component of a pharmaceutical formulation that does not itself induce the production of antibodies in recipients, that is well-tolerated in a subject, and that does not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers, diluents or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0119] The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein, unless otherwise indicated. In certain embodiments, the individual or subject is a human.

[0120] "Administering" or "administration," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0121] The term "sample" refers to a collection (e.g., fluid, cell, or tissue) isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum and serosal fluids, plasma, lymphatic fluid, urine, saliva, cyst fluid, lacrimal fluid, fecal matter, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, e.g., culture media (including conditioned media), lavage fluids, and the like, tissue biopsy samples, fine needle aspirates, surgically removed tissues, organ cultures, or cell cultures.

[0122] "Treatment" and "treating" (and grammatical variations thereof) refer to clinical intervention by which an individual's clinical status is modified. The desired effect of treatment includes, but is not limited to, preventing or delaying the onset of a disease or its recurrence, alleviating symptoms, lessening / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, ameliorating or lessening the disease state, and improved prognosis. In some embodiments, the antibodies of the disclosure are used to delay development of a disease or to slow the progression of a disease.

[0123] An "effective amount" is generally an amount that is sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or their underlying cause, prevent symptoms and / or their underlying cause from occurring, and / or ameliorate or improve damage caused or associated with a disease state (e.g., a lung disease). In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0124] A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptoms, especially states or symptoms associated with the disease state, or otherwise to prevent, hinder, delay, or reverse the progression of the disease state or any other undesirable symptoms associated with the disease in any way. A "prophylactically effective amount" is an amount that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset of the disease state (or recurrence), or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete treatment or prevention need not occur in order for a dose to be considered therapeutically or prophylactically effective. Thus, a therapeutically or prophylactically effective amount can be administered in one or more doses. "Therapeutically effective amount" and "prophylactically effective amount" can vary depending on factors such as the disease state of the individual, the age, gender, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, e.g., improved health status of the patient.

[0125] Antibody Structure

[0126] In certain embodiments, the antibodies provided herein are full-length antibodies.

[0127] In certain embodiments, the antibodies provided herein are antibody fragments.

[0128] In some embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. "Fab" is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. "Fab fragments" can be produced by the proteolytic cleavage of antibodies. "Fab'" contains the VL, CL, and VH and CH1 domains, and also contains the region between the CH1 and CH2 domains, so that the two heavy chains can form a disulfide bond, allowing the formation of F(ab')2 molecules. "Fab'-SH" is a Fab' fragment in which the cysteine residue of the constant region has a free thiol group. "F(ab')2" comprises two Fab fragments that are linked by disulfide bonds in the hinge region.

[0129] In some embodiments, the antibody fragment is a diabody, triabody, or tetrabody. Diabodies are antibody fragments that have two antigen binding sites, comprising linked VH and VL in the same polypeptide chain (VH-VL). By using a linker that is too short to allow for pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, resulting in two antigen binding sites, which can be identical or different

[0130] In some embodiments, the antibody fragment is a single chain Fab fragment. A "single chain Fab fragment" or "scFab" is a polypeptide consisting of a VH, a CH1, a VL, a CL, and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide of at least 30 amino acids. In some embodiments, the linker is a polypeptide of between 32 and 50 amino acids. The single chain Fab fragments are stabilized via the native disulfide bond between CL and CH1. In addition, these single chain Fab molecules can be further stabilized by creating an interchain disulfide bond by the insertion of a cysteine residue, for example at position 44 in the heavy chain variable region and at position 100 in the light chain variable region, according to Kabat numbering.

[0131] In some embodiments, the antibody fragment is a Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0132] In some embodiments, the antibody fragment is a single chain variable fragment (scFv). A "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked in tandem by a short flexible peptide linker, capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically indicated otherwise, a scFv herein can have the VL and VH variable regions in either order, e.g., a scFv can comprise VL-linker-VH or can comprise VH-linker-VL, with respect to the N- and C-termini of the polypeptide.

[0133] In some embodiments, the antibody fragment is a dsFv, which is obtained by linking a polypeptide in which one amino acid residue in each of the VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to known methods.

[0134] In some embodiments, the antibody fragment is a single domain antibody (dAb). A single domain antibody is an antibody fragment comprising the entire or partial heavy chain variable domain or the entire or partial light chain variable domain of an antibody.

[0135] In certain embodiments, an antibody provided herein is a chimeric antibody. In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody.

[0136] In certain embodiments, an antibody is a humanized antibody. Typically, a non- human antibody is humanized by grafting to render it less immunogenic in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable regions in which CDRs or portions thereof are derived from a non-human antibody, while FRs or portions thereof are derived from a human antibody. Optionally, a humanized antibody will also comprise a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody providing the CDR sequences).

[0137] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

[0138] Human framework regions that can be used include, but are not limited to, framework regions selected from "best-fit" approach (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions generated using a design philosophy called "strain improvement" (see, e.g., Schier et al. Gene, 169: 147-155 (1996)).

[0139] Variants of Anti-KIT Antibodies

[0140] In certain embodiments, amino acid sequence variants of the anti-KIT antibodies provided herein are contemplated. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequences of the anti-KIT antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding properties.

[0141] Substitution, Insertion, and Deletion Variants

[0142] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0143] According to common practice, amino acids can be grouped as follows:

[0144] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

[0145] (2) neutral, hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0146] (3) acidic: Asp, Glu;

[0147] (4) basic: His, Lys, Arg;

[0148] (5) small: Gly, Ser, Asn, Gin, Pro;

[0149] (6) aromatic: Trp, Tyr, Phe.

[0150] Non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.

[0151] One type of substitutional variant involves substituting one or more CDRL residues in a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have some alteration (e.g., improvement) in certain biological properties relative to the parent antibody from which they were derived. One example of a substitutional variant is an affinity matured antibody, which can be conveniently generated, e.g., using phage display-based affinity maturation techniques (such as those described herein). Briefly, one or more CDRL residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity). Changes (e.g., substitutions) can be made to CDRs, e.g., to improve antibody affinity. Such changes can be made, e.g., to CDR "hotspot" residues, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and / or residues that contact antigen, while the resulting variant VHand / or VLis tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves CDR-directed approaches, in which a few CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, e.g., using alanine scanning mutagenesis or modeling. In particular, HCDR3 and LCDR3 are often targeted.

[0152] In certain embodiments, substitutions, insertions, or deletions can occur within one or more CDRs so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in a CDR. Such alterations can be outside of antigen contacting residues in a CDR. In certain embodiments of the variant VHand VLsequences provided above, each CDR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.

[0153] One method for identifying residues or regions in an antibody that can serve as targets for mutagenesis is called "alanine scanning mutagenesis" In this approach, one or more residues or groups of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions can be introduced at the amino acid positions which display functional sensitivity to the initial substitutions. Additionally, contact points between the antibody and antigen can be identified through the study of crystal structures of antigen-antibody complexes. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0154] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0155] Recombinant methods

[0156] Anti-KIT antibodies can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding an anti-KIT antibody are provided.

[0157] In some embodiments, the present disclosure provides an isolated nucleic acid encoding an anti-KIT antibody as previously described. Such a nucleic acid can be provided in a separate nucleic acid encoding any of the polypeptide chains previously described. In another aspect, the present disclosure provides one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In another aspect, the present disclosure provides a host cell comprising such a nucleic acid. In some embodiments, a method of producing a polypeptide or fusion protein is provided, wherein the method comprises, culturing a host cell comprising a nucleic acid encoding the polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering the anti-KIT antibody from the host cell (or host cell culture medium).

[0158] For recombinant production of an anti-KIT antibody, nucleic acids encoding the protein are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or obtained through chemical synthesis.

[0159] Suitable host cells for cloning or expression of vector encoding an anti-KIT antibody include prokaryotic or eukaryotic cells described herein. For example, production in bacteria is possible, particularly when glycosylation and Fc effector functions are not needed. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction, and can be further purified.

[0160] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expression of fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells; plant cell cultures also can be utilized as hosts, e.g., US5959177, US6040498, US6420548, US7125978, and US6417429; vertebrate cells can also be used as hosts, e.g., mammalian cell lines adapted to grow in suspension cell culture. Other examples of appropriate mammalian host cell lines are monkey kidney CVl line (COS-7) transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production of antibodies, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0161] Assay

[0162] The anti-KIT antibodies provided herein can be identified, screened, or characterized for their physical / chemical characteristics and / or biological activities by a variety of assays known in the art. In some embodiments, the anti-KIT antibodies of the disclosure are tested for activity, e.g., by known methods such as ELISA, Western blotting, etc.

[0163] Methods of treatment and routes of administration

[0164] Any of the anti-KIT antibodies provided by the disclosure can be used in a method of treatment. In some embodiments, the anti-KIT antibodies provided by the disclosure are used in the manufacture or preparation of a medicament. In some embodiments, the disease is a KIT-associated disease or disorder.

[0165] In some embodiments, a pharmaceutical composition comprising the anti-KIT antibodies is provided, e.g., for any of the pharmaceutical uses or methods of treatment described above. In some embodiments, the pharmaceutical composition comprises any of the anti-KIT antibodies provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0166] The anti-KIT antibodies of the disclosure can be used alone or in combination with other agents for treatment. For example, the antibodies of the disclosure can be co-administered with at least one additional therapeutic agent.

[0167] The anti-KIT antibodies of the disclosure (and any additional therapeutic agents) can be administered by any appropriate means, including parenterally, intrapulmonary, and intranasally, and, if local treatment is required, intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short term or long term. A variety of dosing schedules are contemplated herein, including but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0168] The anti-KIT antibodies of the disclosure will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The anti-KIT antibodies can be formulated with or without carriers and / or diluents and / or adjuvants depending on the route of administration and the type of composition desired. An effective amount of such other agents depends on the amount of anti-KIT antibody present in the pharmaceutical composition, the type and severity of the disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with or without adjuvants as the anti-KIT antibodies described herein, or used in other dosages and / or with or without adjuvants, by

[0169] For the prevention or treatment of disease, the appropriate dosage of an anti-KIT antibody of the present disclosure, when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of treatment molecule, the severity and course of the disease, whether the purpose is preventive or therapeutic, previous therapy, the patient's clinical history and response to the treatment molecule, and the judgment of the treating physician. The treatment molecule is appropriately administered to the patient at one time or over a series of treatments.

[0170] Articles of manufacture

[0171] In another aspect of the present disclosure, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture contains a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-KIT antibody of the present disclosure. The label or package insert indicates that the composition is used for treating the condition of choice. Furthermore, the article of manufacture can further comprise: (a) a first container wherein said composition is contained, said composition comprising an anti-KIT antibody of the present disclosure; and (b) a second container wherein said composition comprising an additional cytotoxic or otherwise therapeutic agent is contained. The article of manufacture in this embodiment of the present disclosure can further comprise a package insert indicating that the compositions can be used to treat the particular condition. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, which can be stored for later combination with the composition contained in the first container. It will be appreciated that the composition of containers of the article of manufacture, and the contents of the containers, can be determined in light of

[0172] Anti-KIT antibodies of the present disclosure

[0173] The present disclosure provides anti-KIT antibodies that have a number of advantageous properties, such as good affinity, therapeutic activity, safety, pharmacokinetic properties, and drugability.

[0174] Exemplary anti-KIT antibodies

[0175] The present disclosure provides an anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0176] I, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 36, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 37, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; or

[0177] II, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 5, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; or

[0178] III, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0179] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, comprising a heavy chain variable region and a light chain variable region, wherein:

[0180] I, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 26, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27; or

[0181] II, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 14, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15; or

[0182] III, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 32, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 33; or

[0183] IV, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 16, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 17.

[0184] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region that is a human IgGl heavy chain constant region variant and a light chain constant region that is a human kappa or lambda light chain constant region; preferably, the human IgGl heavy chain constant region variant comprises mutations L234A, L235A, G237A, M428L and N434S, and the light chain constant region is a human kappa light chain constant region.

[0185] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region that comprises an amino acid sequence as set forth in SEQ ID NO: 18 and a light chain constant region that comprises an amino acid sequence as set forth in SEQ ID NO: 19.

[0186] In some embodiments, the anti-KIT antibody of any of the preceding embodiments, wherein the anti-KIT antibody comprises a heavy chain and a light chain, wherein:

[0187] I, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 29; or

[0188] II, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 20, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 21; or

[0189] III, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 34, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35; or

[0190] IV, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 22, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 23.

[0191] In another aspect, the present disclosure provides use of the anti-KIT antibody of any of the above embodiments in the manufacture of a medicament for preventing or treating urticaria; in particular, use in the manufacture of a medicament for treating and preventing chronic urticaria.

[0192] In some embodiments, the chronic urticaria is selected from the group consisting of chronic spontaneous urticaria, chronic inducible urticaria, and cold contact urticaria.

[0193] DETAILED DESCRIPTION

[0194] Examples

[0195] The following examples and test examples further describe the present application and are not to be construed as limiting the scope of the application. The examples or test examples of the present application do not include detailed description of conventional methods, experimental methods without specific conditions, which are usually carried out according to the conventional conditions, because such methods are well known to those having ordinary skill in the art and are described in many publications, such as Molecular Cloning (Green M R, Sambrook J. Molecular cloning [J]. A Laboratory Manual 4th, 2012.) published by Cold Spring Harbor Laboratory, or Antibody engineering: methods and protocols (Antibody engineering: methods and protocols [M]. Humana Press, 2018.) published by Springer protocols, or according to the conditions recommended by the manufacturer of the experimental materials. Certain experimental materials without specific sources are obtained from the market.

[0196] Example 1. Preparation and screening of anti-KIT hybridoma monoclonal antibodies

[0197] The present disclosure prepared monoclonal antibodies targeting human KIT by hybridoma technology. Human KIT extracellular segment recombinant protein (SEQ ID NO: 1) or M07e cells (purchased from Peking Union Medical College) were used as antigens, Gold Adjuvant (Sigma, T2684) or Alum (Thermo, 77161) as immunoadjuvant, the complex was prepared with antigen and different immunoadjuvants, and the mice were immunized alternately. After the primary and booster immunization, the mice with high antibody titer in serum were selected, and the mouse spleen B cells were collected and fused with myeloma cells to prepare hybridoma cells.

[0198] The corresponding gene was constructed into an expression vector by conventional molecular cloning means, and human KIT extracellular segment recombinant protein (SEQ ID NO: 1) was obtained by transient expression in 293 cells and purified by Ni-NTA affinity chromatography. The amino acid sequence is as follows:

[0199] > Human KIT extracellular segment recombinant protein:

[0200] According to the growth of monoclonal hybridoma cells, the cell culture supernatant was taken for detection. The hybridoma monoclonal antibodies capable of effectively binding to human KIT-ECD (ACROBiosystems, item number: CD7-H52H4) and cynomolgus monkey KIT-ECD (ACROBiosystems, item number: CD7-C52H9) were screened by ELISA, the hybridoma monoclonal antibodies capable of effectively binding to M07e cells were screened by flow cytometry, and the monoclonal antibodies capable of effectively inhibiting the proliferation of M07e cells were screened by biological activity analysis. Through the above-mentioned multiple rounds of screening, the murine monoclonal anti-KIT antibodies 531 and 4A11 with strong binding capacity and good functional activity were obtained. The corresponding monoclonal hybridoma cell strains were expanded, and the cells in the logarithmic growth phase were collected, and the RNA was extracted from the cells by TRIzol (Thermo Fisher Scientific), and the cDNA was obtained by reverse transcription of the RNA with PrimeScript TM Reverse Transcriptase (Takara, 2680A), the cDNA was amplified by PCR using the specific primer combination reported in the literature (the relevant primer sequences are from Antibody Engineering Volume 1, 2nd Edition, page 323, the authors of the book are Roland Kontermann and Stefan Dübel), the PCR products were collected and sequenced, and the obtained nucleotide sequence was translated into the amino acid sequence of the variable region of the murine antibody. The amino acid sequences of the CDR and variable region of the preferred monoclonal hybridoma cell strain are as follows:

[0201] The CDR region sequences of the heavy chain and light chain of the murine anti-KIT antibodies 531 and 4A11 are shown in Table 1 below:

[0202] Table 1. CDR sequences of murine anti-KIT antibodies

[0203] Note: The complementarity determining region (CDR) and framework region (FR) of the variable region of the heavy chain and light chain of each antibody are determined according to the Kabat numbering scheme.

[0204] The heavy chain variable region is denoted as VH, and the light chain variable region is denoted as VL. The VH / VL amino acid sequences of the above-mentioned murine antibodies are as follows:

[0205] > Murine monoclonal antibody 531-VH amino acid sequence

[0206] > Murine monoclonal antibody 531-VL amino acid sequence

[0207] Mouse mAb 4A11-VH amino acid sequence

[0208] Mouse mAb 4A11-VL amino acid sequence

[0209] Note: The primary structure of the above sequences is FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4, the underlined sequences are CDRs determined according to the numbering scheme of Kabat, and the non-underlined sequences are FRs.

[0210] The heavy chain variable region and the light chain variable region of mouse 531 and mouse 4A11 anti-KIT antibodies were recombined with the heavy chain constant region IgG1-AAA+LS and the light chain constant region human kappa chain CL, respectively, to obtain full-length chimeric antibodies 531-Chi and 4A11-Chi. IgG1-AAA+LS comprises L234A+L235A+G237A mutations (abbreviated as AAA) and M428L+N434S mutations (abbreviated as LS mutation combination).

[0211] IgG1-AAA+LS amino acid sequence:

[0212] Human kappa chain CL amino acid sequence:

[0213] 531-Chi heavy chain:

[0214] 531-Chi light chain:

[0215] 4A11-Chi heavy chain:

[0216] 4A11-Chi light chain:

[0217] Note: The underlined sequences are CDRs, and the italic sequences are light chain constant regions or heavy chain constant regions.

[0218] Example 2. Humanization of anti-KIT antibodies of the present application

[0219] 2.1. Humanization of 531 antibody

[0220] The murine 531-VH and VL were compared with the NCBI human antibody variable region germline gene database using IgBlast tool, and IGHV4-59*01 and IGKV3-11*01 were selected as the templates for humanization of 531-VH and VL respectively by homology analysis. The CDRs of the murine 531-VH and VL were grafted into the corresponding human templates, and WGQGTLVTVSS (SEQ ID NO: 24) and FGQGTKVEIK (SEQ ID NO: 25) were selected as FR4 of VH and VL respectively to form CDR-grafted VH and VL with the region structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0221] Here, the amino acid residues of the CDR-grafted VH and VL were subjected to back mutation (the process of mutating the amino acid residues in the CDR-grafted VH and VL that are important for maintaining the correct conformation and affinity of VH and VL into the amino acid residues at the corresponding positions of the murine antibody is called back mutation) or other mutations (mutations and substitutions of specific amino acid residues in CDR / FR to improve the physicochemical properties of the antibody or to reduce the risk of immunogenicity). Preferably, the amino acid residues at positions 1, 37, 48, 67, 71 and 73 of the CDR-grafted VH were mutated, and the specific mutation combination was Q1E, I37V, I48L, V67L, V71R and T73N, to obtain the humanized VH. Preferably, the amino acid residues at positions 24, 36, 47, 56 and 58 of the CDR-grafted VL were mutated, and the specific mutation combination was S24R, Y36F, L47W, S56D and I58V, to obtain the humanized VL. In the process of humanization of the antibody, the positions of the VH and VL amino acid residues were determined using the Kabat coding scheme, Q1E means mutating Q at position 1 to E, and the meanings of other mutations are sequentially similar.

[0222] The sequences of the humanized 531-VH and VL are as follows:

[0223] >531-Hu-VH

[0224] >531-Hu-VL

[0225] Note: In the above sequences, the order of each region of VH and VL is FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4. The part without underlining is FR, the part underlined is CDR determined according to the Kabat coding scheme, and the part double underlined and bolded indicates back mutation or other mutation.

[0226] The above humanized VH and VL were recombined with heavy chain constant region IgG1-AAA+LS (SEQ ID NO: 18) and light chain constant region CL (SEQ ID NO: 19) respectively to obtain the complete 531 humanized antibody 531-Hu.

[0227] The full-length sequence of 531 humanized antibody (denoted as 531-Hu) is as follows:

[0228] > Heavy chain amino acid sequence of 531-Hu humanized antibody

[0229] > Light chain amino acid sequence of 531-Hu humanized antibody

[0230] Note: In the above sequences, underlined part is CDR determined according to Kabat coding scheme, and underlined part is antibody constant region.

[0231] 2.2. Humanization of 4A11 antibody

[0232] Using IgBlast tool, murine 4A11-VH and VL were compared with NCBI human antibody variable region germline gene database respectively, and through homology analysis, IGHV1-46*01 and IGKV6-21*01 were selected as the templates for humanization of 4A11-VH and VL respectively. The CDRs of murine 4A11-VH and VL were transplanted into the corresponding human templates respectively, and WGQGTTVTVSS (SEQ ID NO: 30) and FGGGTKLEIK (SEQ ID NO: 31) were selected as FR4 of VH and VL respectively to form CDR-grafted VH and VL with the region structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0233] Here, the amino acid residues of CDR-grafted VH and VL were subjected to back mutation or other mutations. Preferably, the amino acid residues at positions 1, 48, 49, 67, 69, 71, 73 and 78 of CDR-grafted VH were subjected to back mutation or other mutations, and the specific mutation combination was Q1E, M48I, G49A, V67A, M69L, R71A, T73K and V78A to obtain humanized VH. Preferably, the amino acid residue at position 58 of CDR-grafted VL was subjected to back mutation or other mutations, and the specific mutation was V58I to obtain humanized VL.

[0234] The sequences of 4A11 humanized VH and VL are as follows:

[0235] >4A11-Hu-VH

[0236] >4A11-Hu-VL

[0237] Note: In the above sequences, the order of the regions of VH and VL is FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4. The underlined part is FR, the underlined part is CDR determined according to Kabat coding scheme, and the double underlined and bold part represents back mutation or other mutation.

[0238] The above humanized VH and VL are respectively recombined with heavy chain constant region IgG1-AAA+LS (SEQ ID NO: 18) and light chain constant region CL (SEQ ID NO: 19) to obtain complete 4A11 humanized antibody 4A11-Hu.

[0239] The full-length sequence of 4A11 humanized antibody (denoted as 4A11-Hu) is as follows:

[0240] >4A11-Hu humanized antibody heavy chain amino acid sequence

[0241] >4A11-Hu humanized antibody light chain amino acid sequence

[0242] Note: In the above sequences, the underlined part is CDR determined according to Kabat coding scheme, and the underlined part is the constant region of the antibody.

[0243] Table 2. CDR sequences of humanized antibodies

[0244] Note: The CDR and FR of each antibody heavy chain and light chain variable region are determined according to Kabat numbering scheme.

[0245] The positive control molecule used in the present disclosure is CDX-0159 (Barzolvolimab). The heavy chain and light chain amino acid sequences of Barzolvolimab are derived from WHO Drug Information, Vol. 36, No. 1, 2022.

[0246] >Barzolvolimab heavy chain amino acid sequence:

[0247] >Barzolvolimab light chain amino acid sequence:

[0248] Note: In the above sequences, underlined are CDRs determined according to the Kabat coding scheme, and the underlined part is the constant region of the antibody.

[0249] DNA encoding the above amino acid sequences was synthesized by Suzhou Juyizhi Biotechnology Co., Ltd. or Shanghai Sunny Biotech Co., Ltd., and the above heavy chain and light chain genes were cloned into the expression vector pcDNA3.4 by a conventional method. The corresponding heavy chain and light chain expression vectors were simultaneously transfected into Expi293F cells (Thermo Fisher Scientific, Cat No: A14527) using PEI (Polyethylenimine) to express the antibody. TM The 293F cells were cultured in serum-free medium for 5-6 days, and the cell supernatant was collected and the antibody was purified by Protein A affinity chromatography.

[0250] The purification steps are described as follows: high-speed centrifugation was used to remove impurities in the cell culture supernatant, and the antibody in the supernatant was captured by MabSelect SuRe (Cytiva, Cat No: 17543801) affinity chromatography. First, the MabSelect Sure affinity column was washed with 0.2M NaOH, then washed with pure water and equilibrated with PBS, and the supernatant was passed through the affinity column, and the affinity column was washed with PBS until A280 dropped to the baseline, and the target protein was eluted with 0.1M acetic acid buffer at pH 3.5, and the antibody solution was neutralized with 1M Tris-HCl (pH 8.0). The antibody solution was appropriately concentrated by ultrafiltration, and then the antibody was further purified by gel chromatography column HiLoad Superdex 200 (Cytiva, Cat No: 28989335). The antibody concentration was determined by ultraviolet spectrophotometry, filtered to remove bacteria, and stored in the refrigerator (4°C). This method is used to purify related monoclonal antibodies, and this method can also be used to purify other antibodies or recombinant proteins in the present disclosure.

[0251] Test Example

[0252] Test Example 1: ELISA detection of the binding ability of humanized anti-KIT antibody to KIT

[0253] The ELISA detection steps used in this example are described as follows. Human KIT-ECD (ACROBiosystems, Cat#: CD7-H52H4) was diluted with sodium carbonate buffer (pH 9.0) to 1 pg / mL, and the solution was transferred into a 96-well microplate (100 pL per well) and incubated at 4°C overnight. After washing the plate, phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20 was transferred into the microplate (200 pL per well), and the microplate was incubated at room temperature for 1 hour to block the microplate. The antibody to be tested was gradient-diluted and transferred into the above microplate (100 pL per well), and incubated at room temperature for 1 hour. After washing the plate, a moderately diluted HRP-goat anti-human Fc secondary antibody (Jackson, Cat#: 109-035-088) was added (100 pL per well), and incubated at room temperature for half an hour. After washing the plate, a color developing solution with TMB (3,3',5,5'-Tetramethylbenzidine) as a substrate (KPL, Cat#: 5120-0077) was added (100 pL per well), and incubated at room temperature for 1-5 minutes. When the color developed to an appropriate degree, 50 pL of 2M H2SO4 solution was added to each well to stop the reaction. The OD450 was measured by a microplate reader (Molecular Devices). Data analysis was performed by GraphPad Prism 10. The results are shown in Table 3.

[0254] Table 3. Binding ability of anti-KIT antibodies to human KIT recombinant protein

[0255] The experimental results show that the humanized antibodies 531-Hu and 4A11-Hu have good binding ability to human KIT recombinant protein as compared with the corresponding chimeric antibodies.

[0256] Test Example 2, Biacore detection of the affinity of anti-KIT antibodies to KIT

[0257] In this example, the extracellular segment of KIT recombinant proteins from human, cynomolgus monkey and dog are denoted as human KIT-ECD (ACROBiosystems, Cat#: CD7-H52H4), cynomolgus monkey KIT-ECD (ACROBiosystems, Cat#: CD7-C52H9) and dog KIT-ECD

sequence from UniProt (Entry: O97799), truncated 28-527 amino acid sequence, signal peptide added at N-terminus, 6*His tag added at C-terminus, then construct expression vector using conventional molecular cloning method, express recombinant protein in HEK293 cells by transient transfection, then purify by Ni-NTA affinity chromatography

[0258] Table 4. Binding, dissociation and equilibrium dissociation constants of anti-KIT humanized antibodies to KIT

[0259] The experimental results show that the humanized antibodies 531-Hu and 4A11-Hu of the disclosure have high affinity to KIT recombinant proteins from multiple species.

[0260] Test Example 3, FACS detection of the binding of anti-KIT antibodies to cell surface KIT

[0261] The binding ability of the humanized antibodies of the present disclosure to M07e cells was tested by FACS. M07e cell culture was collected, centrifuged at 300g for 5 minutes to collect the cells, washed the cells twice with PBS containing 0.5% BSA and resuspended, after counting, 5pL of Fc receptor blocking solution (BioLegend, Cat# 422303) was added per 1 million cells, 100,000 cells per well were transferred into a round-bottom 96-well plate, and incubated at room temperature for 10 minutes. The humanized antibodies of the present disclosure were gradiently diluted in a 96-well plate with PBS containing 0.5% BSA. The gradiently diluted antibodies were transferred into the above-mentioned round-bottom 96-well plate with M07e cells, and the cells and antibodies were mixed uniformly with a multichannel pipette, and incubated in a 4°C refrigerator for 1 hour. The round-bottom 96-well plate was centrifuged at 300g for 5 minutes, after the supernatant was discarded, the cells were washed twice with PBS, 100pL of Alexa Fluor 488-labeled Goat-anti-human IgG (Thermo Fisher Scientific, Cat# A11013) diluted moderately with PBS containing 1% BSA was added to each well, and incubated at 4°C in the dark for 30 minutes. The round-bottom 96-well plate was centrifuged at 300g for 5 minutes, after the supernatant was discarded, the cells were washed twice with PBS, and the cells were fixed with Fix Buffer I (BD Biosciences, Cat# 557870) (5 minutes at room temperature). The round-bottom 96-well plate was centrifuged at 300g for 5 minutes, after the supernatant was discarded, the cells were washed twice with PBS, and the fluorescence intensity of the cells was measured on an Attune NxT Cytometer. The FACS data were analyzed by Flowjo 10.9, and the data were processed and analyzed by Graphpad Prism 10. The results are shown in Table 5. TM 488-labeled Goat-anti-human IgG (Thermo Fisher Scientific, Cat# A11013), and incubated at 4°C in the dark for 30 minutes. The round-bottom 96-well plate was centrifuged at 300g for 5 minutes, after the supernatant was discarded, the cells were washed twice with PBS, and the cells were fixed with Fix Buffer I (BD Biosciences, Cat# 557870) (5 minutes at room temperature). The round-bottom 96-well plate was centrifuged at 300g for 5 minutes, after the supernatant was discarded, the cells were washed twice with PBS, and the fluorescence intensity of the cells was measured on an Attune NxT Cytometer. The FACS data were analyzed by Flowjo 10.9, and the data were processed and analyzed by Graphpad Prism 10. The results are shown in Table 5.

[0262] Table 5. Binding ability of anti-KIT humanized antibodies to M07e cells

[0263] The experimental results show that the humanized antibodies 531-Hu and 4A11-Hu involved in the present disclosure have high binding ability to M07e cells.

[0264] Test Example 4, Proliferation inhibition activity of anti-KIT antibodies

[0265] Stem cell factor (SCF) as a ligand of KIT, can induce KIT dimerization and downstream signal transduction, stimulate M07e cell proliferation. Anti-KIT antibody can block the downstream signal transduction of KIT induced by SCF, thereby inhibiting the proliferation of M07e cells. The method for determining the proliferation inhibition activity of anti-KIT antibody is described as follows. M07e cells are routinely cultured and passaged according to the supplier's recommended method. M07e cells in the logarithmic growth phase are inoculated into a 96-well plate, 50 μL of cell suspension containing 10,000 cells per well. SCF (PeproTech; Catalog No: 300-07) is diluted to 200 ng / mL with a culture medium containing 2% FBS, and the antibody to be tested is gradiently diluted with the solution, and then the mixed solution of SCF and the antibody is transferred into the 96-well plate inoculated with cells (50 μL per well), and after mixing, the 96-well plate is placed in a carbon dioxide cell incubator for further incubation. After 72 hours, the cell viability is detected by using Luminescent Cell Viability Assay (Promega, Catalog No: G7572, referred to as CTG). The luminescence is measured by using a Microplate reader (PerkinElmer). Data analysis is performed by using GraphPad Prism 10. The results are shown in Table 6.

[0266] Table 6. Proliferation inhibition activity of anti-KIT humanized antibodies on M07e cells

[0267] The experimental results show that the humanized antibodies 531-Hu and 4A11-Hu involved in the disclosure have high proliferation inhibition activity on M07e cells.

[0268] Test Example 5, Inhibition activity of anti-KIT antibodies on induced mast cells

[0269] This example determines the activity of anti-KIT antibodies in inhibiting the release of inflammatory factors by mast cells. In vitro, human peripheral blood-derived CD34 + Hematopoietic stem cells are induced into mature human mast cells, and the maturation process of mast cells is accelerated by adding human low-density lipoprotein (LDL, Absin, Catalog No: abs47014900), and the degree of maturation of mast cells is determined by the expression of KIT and FceRI. An appropriate amount of mature mast cells (96-well plate, 20,000 cells per well) is collected by centrifugation at 300g for 8 minutes, and an experimental culture medium is used [the experimental culture medium is a self-prepared culture medium containing 1x GlutaMAXTM IMDM medium (Thermofisher Scientific, 12440053) with 1X pyruvate sodium (Gibco, 11360070), 50 mM beta-mercaptoethanol (Invitrogen, 21985023), 0.5% bovine serum albumin (Solarbio, A8020), 1X insulin-transferrin-selenium (Gibco, 41400-045), and antibiotics (Gibco, 15240062). After resuspension and washing the cells twice, the cells were resuspended at a concentration of 5 x 105cells / mL, and 100 ng / mL IgE (Sigma-Aldrich, AG30P) was added to the suspension. The cells were incubated overnight in a 37 °C incubator. The next day, the cells were collected by centrifugation, washed twice with IMDM medium, and resuspended at a concentration of 2 x 105cells / mL in experimental medium. The cells were seeded in a 96-well cell culture plate (Thermofisher Scientific, 062096) at a density of 100 μL per well. The anti-KIT antibodies were serially diluted in experimental medium and added to the cell culture and mixed well. The cells were incubated in a 37 °C incubator for 4 hours. Then, 100 ng / mL SCF and anti-IgE (Bethyl Laboratories, A80-109A) were added to the cell culture and incubated in a 37 °C incubator overnight (about 16 hours). The next day, the cell supernatant was collected by centrifugation at 300 g for 8 minutes, and the GM-CSF level in the cell culture supernatant was determined using a GM-CSF ELISA detection kit (R&D, 1117302). The results are shown in FIG. 1A and FIG. 1B. 5 5 The results of the experiments shown in FIG. 1A and FIG. 1B demonstrate that the humanized antibodies 531-Hu and 4A11-Hu of the present disclosure have stronger inhibitory activity on the secretion of GM-CSF by mast cells than the control antibody CDX-0159.

[0270] The results of the experiments shown in FIG. 1A and FIG. 1B demonstrate that the humanized antibodies 531-Hu and 4A11-Hu of the present disclosure have stronger inhibitory activity on the secretion of GM-CSF by mast cells than the control antibody CDX-0159.

[0271] Test Example 6: Pharmacodynamic effects of anti-KIT antibodies on canine skin mast cells

[0272] Two groups of beagle dogs, each group of four, half male and half female, were taken skin tissue samples one week before administration, sectioned and stained, and the number of mast cells was determined. The anti-KIT antibody was administered by single-point subcutaneous injection at the neck, with an administration volume of 0.6 mL / kg and an administration dose of 3 mpk. The day of administration was designated as day 0. After administration, skin tissue was taken on days 7 and 14 and the number of mast cells in the skin was determined. The number of skin mast cells one week before administration was normalized to 100%, and the relative number of mast cells was the percentage of the number of skin mast cells after administration relative to the number of skin mast cells before administration. The results of the experiment are shown in FIG. 2.​

[0273] The results show that, compared with before dosing, CDX-0159 reduced the number of mast cells in the skin of beagle dogs by 4.5% and 8.3% on day 7 and day 14, respectively, and 531-Hu reduced the number of mast cells in the skin of beagle dogs by 39.8% and 50.3% on day 7 and day 14, respectively, and 531-Hu was significantly more effective than the control antibody CDX-0159 in depleting mast cells in the skin. On day 14, there was a significant difference in the pharmacodynamic effect of 531-Hu and CDX-0159 on the number of mast cells (t-test P value of 0.013 < 0.05).

[0274] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be appreciated that certain changes and modifications can be practiced within the scope of the disclosure. The disclosure described herein is intended to be illustrative, and not restrictive, of the scope of the present disclosure. The disclosure contained herein, including the claims, is expressly incorporated by reference in its entirety.

Claims

An anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein: I. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 26, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 27; or II. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 15; or III. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 32, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 33; or IV. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the same amino acid sequence as the heavy chain variable region set forth in SEQ ID NO: 16, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the same amino acid sequence as the light chain variable region set forth in SEQ ID NO: 17; or V. the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 38, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 39, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; wherein SEQ ID NO: 38 is LCDR1 as set forth in X1ASSSVSYMH, wherein X1 is R or S; SEQ ID NO: 39 is LCDR2 as set forth in STSNLAX2, wherein X2 is D or S; or VI. the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:

13. The anti-KIT antibody of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: ​ ​ I, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 36, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 37, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; or II, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 3, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 4; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 5, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7; or III, the HCDR1 of the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 10; the LCDR1 of the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO:

13. The anti-KIT antibody according to claim 1 or 2, wherein the anti-KIT antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; Preferably, the anti-KIT antibody is a murine antibody, a chimeric antibody, or a humanized antibody; More preferably, the anti-KIT antibody is a humanized antibody. The anti-KIT antibody according to any one of claims 1 to 3, wherein: I, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27 or an amino acid sequence having at least 75% sequence identity thereto; or II, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15 or an amino acid sequence having at least 75% sequence identity thereto; or III, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% sequence identity thereto. III, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence that has at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence that has at least 75% sequence identity thereto; or IV, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 16 or an amino acid sequence that has at least 75% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 17 or an amino acid sequence that has at least 75% sequence identity thereto. The anti-KIT antibody according to any one of claims 1 to 4, wherein the anti-KIT antibody is an antibody fragment; preferably, the antibody fragment is selected from a Fab, a Fab', a F(ab')2, a Fv, a scFv, or a dsFv. The anti-KIT antibody according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is derived from a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant region, and the light chain constant region is a human kappa or lambda light chain constant region; more preferably, the heavy chain constant region is derived from a human IgGl heavy chain constant region, and the light chain constant region is a human kappa or lambda light chain constant region; Most preferably, the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18, and the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO:

19. The anti-KIT antibody according to any one of claims 1 to 4, and 6, wherein the anti-KIT antibody comprises a heavy chain and a light chain, wherein: I, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 28 or an amino acid sequence that has at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 29 or an amino acid sequence that has at least 85% sequence identity thereto; or II, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence that has at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 21 or an amino acid sequence that has at least 85% sequence identity thereto; or III, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence that has at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85% sequence identity thereto; or IV, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 22 or an amino acid sequence that has at least 85% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 23 or an amino acid sequence that has at least 85% sequence identity thereto. A pharmaceutical composition comprising the anti-KIT antibody of any one of claims 1 to 7, and one or more pharmaceutically acceptable carriers, diluents or excipients. An isolated nucleic acid encoding the anti-KIT antibody of any one of claims 1 to 7. A host cell comprising the isolated nucleic acid of claim 9. A method of preventing or treating a disease or disorder, the method comprising administering to a subject the anti-KIT antibody of any one of claims 1 to 7, or the pharmaceutical composition of claim 8; wherein the disease or disorder is selected from the group consisting of inflammatory diseases, eye diseases, cancers, blood diseases, allergies and autoimmune diseases; Preferably, the disease or disorder is selected from the group consisting of nodular prurigo, esophagitis, asthma, atopic dermatitis, urticaria, myeloma, connective tissue tumor, leukemia, lung cancer, gastrointestinal stromal tumor, hemoglobinopathy, retinal disorder, diabetic macular edema and wet age-related macular degeneration; More preferably, the hemoglobinopathy is selected from the group consisting of sickle cell disease and beta-thalassemia; the urticaria is selected from the group consisting of chronic urticaria, chronic spontaneous urticaria, chronic inducible urticaria and cold contact urticaria; the lung cancer is small cell lung cancer; the leukemia is chronic myelocytic leukemia or acute myeloid leukemia; the esophagitis is eosinophilic esophagitis.

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