Anti-KIT antibodies and uses thereof

Abstract

Provided herein are antibodies that immunospecifically bind to the receptor tyrosine kinase, KIT, and uses thereof. Also provided are polynucleotides and vectors encoding such antibodies, cells containing such polynucleotides or vectors, and methods of making such antibodies.

Description

[Technical field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 140,642, filed January 22, 2021, and U.S. Provisional Application No. 63 / 238,649, filed August 30, 2021, which are incorporated by reference in their entireties.

[0002] (Reference to electronically submitted sequence listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "Seqlisting_12638-170-228.txt", created on January 10, 2022, and having a size of 44,171 bytes.

[0003] (1. Field) Provided herein are antibodies that immunospecifically bind to the receptor tyrosine kinase, KIT, and uses thereof. Also provided are polynucleotides and vectors encoding such antibodies, cells containing such polynucleotides or vectors, and methods of making such antibodies. [Background technology]

[0004] (2.Background) KIT (or c-Kit) is a type III receptor tyrosine kinase encoded by the c-kit gene. KIT contains five extracellular immunoglobulin (Ig)-like domains, a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (see, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464). The human c-kit gene encoding the KIT receptor was cloned as described in Yarden et al., EMBO J., 1987, 6:3341-3351. KIT is also known as CD117 or stem cell factor receptor ("SCFR"), since it is the receptor for stem cell factor ("SCF") ligand (also known as Steel factor or Kit ligand). Binding of SCF ligand to the first three extracellular Ig-like domains of KIT induces receptor dimerization, thereby activating intrinsic tyrosine kinase activity through phosphorylation of specific tyrosine residues in the juxtamembrane domain and kinase domain (see, e.g., Weiss and Schlessinger, Cell, 1998, 94:277-280; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464). Members of the Stat, Src, ERK, and AKT signaling pathways have been shown to be downstream signal transducers of KIT signaling.

[0005] The fourth (D4) and fifth (D5) extracellular Ig-like domains of KIT are believed to mediate receptor dimerization (see, e.g., International Patent Application Publication No. WO 2008 / 153926; Yuzawa et al., Cell, 2007, 130:323-334).

[0006] Expression of KIT has been detected in a variety of cell types, including mast cells, stem cells, brain cells, melanoblasts, ovarian cells, and cancer cells (e.g., leukemia cells) (see, e.g., Besmer, P., Curr. Opin. Cell Biol., 1991, 3:939-946; Lyman et al., Blood, 1998, 91:1101-1134; Ashman, LK, Int. J. Biochem. Cell Biol., 1999, 31:1037-1051; Kitamura et al., Mutat. Res., 2001, 477:165-171; Mol et al., J. Biol. Chem., 2003, 278:31461-31464). In addition, KIT plays an important role in hematopoiesis, melanogenesis, and gametogenesis (see Ueda et al., Blood, 2002, 99:3342-3349).

[0007] Antibodies that immunospecifically bind to human KIT are known, for example, from International Patent Publication WO2014018625A1, which is incorporated herein by reference in its entirety.

[0008] There is a need to provide improved antibodies against human KIT. Summary of the Invention

[0009] (3. Overview) In one aspect, provided herein is an antibody that immunospecifically binds to human KIT, comprising: (i) a light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; (ii) a heavy chain variable region ("VH") comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising unnatural amino acids 234A, 235Q, and 322Q, as numbered by the EU index in Kabat. In a specific embodiment, the modified (e.g., mutated) human IgG1 Fc region or domain further comprises unnatural amino acids 252Y, 254T, and 256E, as numbered by the EU index in Kabat.

[0010] In a specific embodiment, (i) the VL of the antibody has the amino acid sequence: [ka] (where X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K3 is an amino acid having an aliphatic hydroxyl side chain, and X K4 is an amino acid having an aliphatic hydroxyl side chain or is P; X K5 is an amino acid having a charged or acidic side chain, and X K6 is an amino acid having an aromatic side chain; (ii) the VH of the antibody comprises the amino acid sequence: [ka] (where X H1 is an amino acid having an aliphatic side chain, and X H2 is an amino acid having an aliphatic side chain, and X H3 is an amino acid with a polar or basic side chain, and XH4 is an amino acid having an aliphatic side chain, and X H5 is an amino acid having an aliphatic side chain, and X H6 is an amino acid with an acidic side chain, and X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain) In a specific embodiment, X K1 is the amino acid F or S, and X K2 is the amino acid A or S, and X K3 is the amino acid T or S, and X K4 is the amino acid S or P, and X K5 is the amino acid D or T, and X K6 is an amino acid F or Y, and X H1 is an amino acid L or V, and X H2 is an amino acid L or V, and X H3 is the amino acid K or R, and X H4 is an amino acid V or A, and X H5 is an amino acid L or I, and X H6 is the amino acid E or D, and X H7 is an amino acid Q or E, and X H8 is the amino acid S or T.

[0011] In specific embodiments, (i) the VL of the antibody comprises the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16, and (ii) the VH of the antibody comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11 or 12.

[0012] In a specific embodiment, the antibody comprises: (i) a VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising the non-natural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference.

[0013] In a specific embodiment, the antibody comprises: (i) a VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising the unnatural amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

[0014] In a specific embodiment, the antibody has the amino acid sequence: [ka] The heavy chain comprises:

[0015] In a specific embodiment, the antibody has the amino acid sequence: [ka] The light chain comprises

[0016] In a specific embodiment, the antibody has (i) the amino acid sequence: [ka] and (ii) a heavy chain comprising the amino acid sequence: [ka] The light chain comprises

[0017] In another aspect, provided herein is a conjugate comprising an antibody provided herein linked to an agent.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising an antibody or conjugate provided herein and a pharma- ceutically acceptable carrier.

[0019] In another aspect, provided herein is an antibody provided herein, or a polynucleotide or combination of polynucleotides comprising a nucleotide sequence encoding the VH and VL of said antibody.

[0020] In another aspect, provided herein is a vector or combination of vectors comprising a polynucleotide or combination of polynucleotides provided herein.

[0021] In another aspect, provided herein is a host cell comprising a vector or combination of vectors provided herein, or a polynucleotide or combination of polynucleotides provided herein.

[0022] In another aspect, provided herein is a kit comprising an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein.

[0023] In another aspect, provided herein is a method for preventing, treating or managing KIT-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein.In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, an allergic inflammation, an inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder.In a particular embodiment, the mast cell-associated disorder is chronic urticaria.In one embodiment, the chronic urticaria is chronically triggered urticaria.In one embodiment, the chronically triggered urticaria is cold urticaria.In one embodiment, the chronically triggered urticaria is symptomatic dermatographia.In one embodiment, the chronically triggered urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic spontaneous urticaria. In a specific embodiment, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

[0024] In some embodiments, the method provided herein further comprises administering to the subject a second therapeutic agent.In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent.

[0025] In another aspect, provided herein is a method for inhibiting KIT activity in a cell expressing KIT, comprising contacting the cell with an effective amount of an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein. In a specific embodiment, the method inhibits KIT activity in a cell expressing KIT by at least about 10%.

[0026] In another aspect, provided herein is an in vitro method for diagnosing a subject having a KIT-associated disorder, comprising contacting an antibody provided herein with a cell or sample obtained from the subject and detecting the expression level of KIT in the cell or sample.

[0027] In another aspect, provided herein is a method of producing an antibody, comprising culturing a host cell provided herein and / or expressing the antibody using a host cell provided herein. In a specific embodiment, the method further comprises purifying the antibody obtained from the host cell.

[0028] In another aspect, provided herein is the use of the antibody provided herein, the conjugate provided herein, or the pharmaceutical composition provided herein for the manufacture of a medicament for preventing, treating, or managing a KIT-associated disorder.In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder.In a particular embodiment, the mast cell-associated disorder is chronic urticaria.In one embodiment, the chronic urticaria is chronically triggered urticaria.In one embodiment, the chronically triggered urticaria is cold urticaria.In one embodiment, the chronically triggered urticaria is symptomatic dermatographia.In one embodiment, the chronically triggered urticaria is cholinergic urticaria.In another embodiment, the chronic urticaria is chronically spontaneous urticaria. In a specific embodiment, the KIT-associated disorder is an eosinophil-associated disorder, for example, eosinophilic esophagitis (EoE).

[0029] In some embodiments, the medicament described herein is prepared to be administered in combination with a second therapeutic agent.In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent.

[0030] In another aspect, provided herein is the use of an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein for the manufacture of a medicament for inhibiting KIT activity in a cell expressing KIT. In a specific embodiment, the medicament inhibits KIT activity in a cell expressing KIT by at least about 10%.

[0031] In another aspect, provided herein is an antibody as described herein, a conjugate as described herein, or a pharmaceutical composition as described herein for use in a method for preventing, treating, or managing a KIT-associated disorder. In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder. In a particular embodiment, the mast cell-associated disorder is chronic urticaria. In one embodiment, the chronic urticaria is chronically triggered urticaria. In one embodiment, the chronically triggered urticaria is cold urticaria. In one embodiment, the chronically triggered urticaria is symptomatic dermatographia. In one embodiment, the chronically triggered urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic spontaneous urticaria. In a specific embodiment, the KIT-associated disorder is an eosinophil-associated disorder, for example, eosinophilic esophagitis (EoE).

[0032] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent.

[0033] In another aspect, provided herein is an antibody as described herein, a conjugate as described herein, or a pharmaceutical composition as described herein for use in a method of inhibiting KIT activity in a cell expressing KIT. In a specific embodiment, the method inhibits KIT activity in a cell expressing KIT by at least about 10%.

[0034] In another aspect, provided herein is a method of preventing, treating, or managing chronic urticaria in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharma- ceutical acceptable carrier. In one embodiment, human KIT comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the antibody specifically binds to the D4 or D5 region of human KIT. In one embodiment, the antibody comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human Fc region or domain). In one embodiment, the antibody has reduced Fc receptor binding activity (particularly reduced FcγR binding activity). In one embodiment, the antibody does not induce significant degranulation of human mast cells expressing FcgRI. In certain embodiments, the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.In certain embodiments, the antibody comprises: (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In one embodiment, the chronic urticaria is chronic triggered urticaria. In one embodiment, the chronic triggered urticaria is cold urticaria. In one embodiment, the chronic triggered urticaria is symptomatic dermatographia. In one embodiment, the chronic triggered urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic spontaneous urticaria.

[0035] In some embodiments, the method provided herein further comprises administering to the subject a second therapeutic agent.In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent.

[0036] In another aspect, provided herein is the use of (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharma- ceutical acceptable carrier, for the manufacture of a medicament for preventing, treating, or managing chronic urticaria in a subject. In one embodiment, human KIT comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the antibody specifically binds to the D4 or D5 region of human KIT. In one embodiment, the antibody comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human Fc region or domain). In one embodiment, the antibody has reduced Fc receptor binding activity (particularly reduced FcγR binding activity). In one embodiment, the antibody does not induce significant degranulation of human mast cells expressing FcgRI. In one embodiment, the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.In certain embodiments, the antibody comprises: (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In a specific embodiment, the chronic urticaria is chronic triggered urticaria. In one embodiment, the chronic triggered urticaria is cold urticaria. In one embodiment, the chronic triggered urticaria is symptomatic dermatographia. In one embodiment, the chronic triggered urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic spontaneous urticaria.

[0037] In some embodiments, the medicament described herein is prepared to be administered in combination with a second therapeutic agent.In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent.

[0038] In another aspect, provided herein is (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharma- ceutical acceptable carrier, for use in a method for preventing, treating, or managing chronic urticaria. In one embodiment, human KIT comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the antibody specifically binds to the D4 or D5 region of human KIT. In one embodiment, the antibody comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human Fc region or domain). In one embodiment, the antibody has reduced Fc receptor binding activity (particularly reduced FcγR binding activity). In one embodiment, the antibody does not induce significant degranulation of human mast cells expressing FcgRI. In one embodiment, the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.In certain embodiments, the antibody comprises: (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In a specific embodiment, the chronic urticaria is chronic triggered urticaria. In one embodiment, the chronic triggered urticaria is cold urticaria. In one embodiment, the chronic triggered urticaria is symptomatic dermatographia. In one embodiment, the chronic triggered urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic spontaneous urticaria.

[0039] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. 3.1 Exemplary Implementations 1. An antibody that immunospecifically binds to human KIT, (i) a light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; (ii) a heavy chain variable region ("VH") comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain containing the unnatural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference; The antibody comprising: 2. The antibody of embodiment 1, wherein the modified (e.g., mutated) human IgG1 Fc region or domain further comprises the unnatural amino acids 252Y, 254T and 256E, as numbered by the EU index as set forth in the Kabat reference. 3. (i) VL has the amino acid sequence: [ka] (where X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K3 is an amino acid having an aliphatic hydroxyl side chain, and X K4 is an amino acid having an aliphatic hydroxyl side chain or is P; X K5 is an amino acid having a charged or acidic side chain, and X K6 is an amino acid having an aromatic side chain; and (ii) VH has the amino acid sequence: [ka] (where X H1 is an amino acid having an aliphatic side chain, and X H2 is an amino acid having an aliphatic side chain, and X H3 is an amino acid with a polar or basic side chain, and X H4 is an amino acid having an aliphatic side chain, and X H5 is an amino acid having an aliphatic side chain, and X H6 is an amino acid with an acidic side chain, and X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 The antibody of embodiment 1 or 2, comprising: 4. X K1 is an amino acid F or S, and X K2 is an amino acid A or S, and X K3 is the amino acid T or S, and X K4 is the amino acid S or P, and X K5 is the amino acid D or T, and X K6 is an amino acid F or Y, and X H1 is an amino acid L or V, and X H2 is an amino acid L or V, and X H3 is an amino acid K or R, and X H4 is an amino acid V or A, and X H5 is an amino acid L or I, and X H6 is amino acid E or D, and X H7 is an amino acid Q or E, and X H8 The antibody of embodiment 3, wherein is the amino acid S or T. 5. i) the VL comprises the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16; and ii) VH comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11 or 12; An antibody described in any one of embodiments 1 to 4. 6. (i) a VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain containing the unnatural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference; The antibody according to any one of embodiments 1 to 5, comprising: 7. (i) a VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain that contains the unnatural amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index as set forth in the Kabat reference. The antibody according to any one of embodiments 1 to 5, comprising: 8. Amino acid sequence: [ka] The antibody of any one of embodiments 1 to 5, comprising a heavy chain comprising: 9. Amino acid sequence: [ka] The antibody of any one of embodiments 1 to 5, comprising a light chain comprising: 10. Amino acid sequence: [ka] and a heavy chain comprising the amino acid sequence: [ka] The antibody of any one of embodiments 1 to 5, comprising a light chain comprising: 11. A conjugate comprising the antibody according to any one of embodiments 1 to 10 linked to an agent. 12. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 10, or a conjugate according to embodiment 11, and a pharma- ceutical acceptable carrier. 13. An antibody according to any one of embodiments 1 to 10, or a polynucleotide or combination of polynucleotides comprising a nucleotide sequence encoding the VH and VL of said antibody. 14. A vector or a combination of vectors comprising a polynucleotide or a combination of polynucleotides according to embodiment 13. 15. A host cell comprising a vector or combination of vectors according to embodiment 14 or a polynucleotide or combination of polynucleotides according to embodiment 13. 16. A kit comprising an antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12. 17. A method for preventing, treating or managing a KIT-related disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody according to any one of embodiments 1 to 10, the conjugate according to embodiment 11, or the pharmaceutical composition according to embodiment 12. 18. The method of embodiment 17, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. 19. The method of embodiment 18, wherein the KIT-associated disorder is a mast cell-associated disorder. 20. The method of embodiment 19, wherein the mast cell-related disorder is chronic urticaria. 21. The method of embodiment 20, wherein the chronic urticaria is chronic inducible urticaria. 22. The method of embodiment 21, wherein the chronically induced urticaria is cold urticaria. 23. The method of embodiment 21, wherein the chronically induced urticaria is symptomatic dermatographia. 24. The method of embodiment 21, wherein the chronically induced urticaria is cholinergic urticaria. 25. The method of embodiment 20, wherein the chronic urticaria is chronic spontaneous urticaria. 26. The method of embodiment 18, wherein the KIT-associated disorder is an eosinophil-associated disorder. 27. The method of any one of embodiments 17 to 26, further comprising administering to the subject a second therapeutic agent. 28. The method of embodiment 27, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. 29. A method for inhibiting KIT activity in a cell expressing KIT, comprising contacting the cell with an effective amount of an antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12. 30. The method of embodiment 29, wherein the method inhibits KIT activity by at least about 10% in cells expressing KIT. 31. An in vitro method for diagnosing a subject having a KIT-associated disorder, comprising contacting a cell or a sample obtained from the subject with an antibody according to any one of embodiments 1 to 10, and detecting the expression level of KIT in the cell or the sample. 32. A method for producing an antibody, said method comprising culturing a host cell of embodiment 15 and / or expressing the antibody using said host cell. 33. The method of embodiment 32, further comprising purifying the antibody obtained from the host cell. 34. Use of an antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12 for the manufacture of a medicament for preventing, treating or managing a KIT-related disorder. 35. The use according to embodiment 34, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. 36. The use according to embodiment 35, wherein the KIT-associated disorder is a mast cell-associated disorder. 37. The use according to embodiment 36, wherein the mast cell-related disorder is chronic urticaria. 38. The use according to embodiment 37, wherein the chronic urticaria is chronic inducible urticaria. 39. The use according to embodiment 38, wherein the chronically induced urticaria is cold urticaria. 40. The use according to embodiment 38, wherein the chronically induced urticaria is symptomatic dermatographia. 41. The use according to embodiment 38, wherein the chronically induced urticaria is cholinergic urticaria. 42. The use according to embodiment 37, wherein the chronic urticaria is chronic spontaneous urticaria. 43. The use according to embodiment 35, wherein the KIT-associated disorder is an eosinophil-associated disorder. 44. The use according to any one of embodiments 34 to 43, wherein the medicament is prepared to be administered in combination with a second therapeutic agent. 45. The use of embodiment 44, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. 46. ​​Use of an antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12 for the manufacture of a medicament for inhibiting KIT activity in a cell expressing KIT. 47. The use according to embodiment 46, wherein the medicament inhibits KIT activity by at least about 10% in cells expressing KIT. 48. An antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12 for use in a method for preventing, treating or managing a KIT-related disorder. 49. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 48, wherein the KIT-related disorder is a mast cell-related disorder, an eosinophil-related disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. 50. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 49, wherein the KIT-associated disorder is a mast cell-associated disorder. 51. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 50, wherein the mast cell-related disorder is chronic urticaria. 52. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 51, wherein the chronic urticaria is chronic inducible urticaria. 53. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 52, wherein the chronic induced urticaria is cold urticaria. 54. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 52, wherein the chronic induced urticaria is symptomatic dermatographia. 55. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 52, wherein the chronically induced urticaria is cholinergic urticaria. 56. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 51, wherein the chronic urticaria is chronic spontaneous urticaria. 57. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 49, wherein the KIT-associated disorder is an eosinophil-associated disorder. 58. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 48 to 57, wherein the method further comprises administering to the subject a second therapeutic agent. 59. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 58, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. 60. An antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12 for use in a method for inhibiting KIT activity in a cell expressing KIT. 61. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 60, wherein the method inhibits KIT activity by at least about 10% in cells expressing KIT. 62. A method for preventing, treating, or managing chronic urticaria in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharma- ceutical acceptable carrier. 63. The method of embodiment 62, wherein the human KIT comprises the amino acid sequence of SEQ ID NO:1. 64. The method of embodiment 62 or 63, wherein the antibody specifically binds to the D4 or D5 region of human KIT. 65. The method of any one of embodiments 62 to 64, wherein the antibody comprises an altered (e.g., mutated) Fc region or domain. 66. The method of any one of embodiments 62 to 65, wherein the antibody comprises an altered (e.g., mutated) human Fc region or domain. 67. The method of any one of embodiments 62 to 66, wherein the antibody has reduced Fc receptor binding activity (particularly reduced FcγR binding activity). 68. A method according to any one of embodiments 62 to 67, wherein the antibody does not induce significant degranulation of human mast cells expressing FcgRI. 69. A method according to any one of embodiments 62 to 68, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity. 70. The antibody, (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; The method of any one of embodiments 62 to 69, comprising: 71. The method of embodiment 70, wherein the chronic urticaria is chronic inducible urticaria. 72. The method of embodiment 71, wherein the chronically induced urticaria is cold urticaria. 73. The method of embodiment 71, wherein the chronically induced urticaria is symptomatic dermatographia. 74. The method of embodiment 71, wherein the chronically induced urticaria is cholinergic urticaria. 75. The method of embodiment 70, wherein the chronic urticaria is chronic spontaneous urticaria. 76. The method of any one of embodiments 70-75, further comprising administering to the subject a second therapeutic agent. 77. The method of embodiment 76, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. 78. Use of (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharma- ceutical acceptable carrier, for the manufacture of a medicament for preventing, treating, or managing chronic urticaria in a subject. 79. The use described in embodiment 78, wherein the human KIT comprises the amino acid sequence of SEQ ID NO:1. 80. The use of embodiment 78 or 79, wherein the antibody specifically binds to the D4 or D5 region of human KIT. 81. The use of any one of embodiments 78 to 80, wherein the antibody comprises an altered (e.g., mutated) Fc region or domain. 82. The use according to any one of embodiments 78 to 81, wherein the antibody comprises a modified (e.g., mutated) human Fc region or domain. 83. The use according to any one of embodiments 78 to 82, wherein the antibody has reduced Fc receptor binding activity (in particular reduced FcγR binding activity). 84. The use according to any one of embodiments 78 to 83, wherein the antibody does not induce significant degranulation of human mast cells expressing FcgRI. 85. The use according to any one of embodiments 78 to 84, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity. 86. The antibody: (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; The use according to any one of embodiments 78 to 85, comprising: 87. The use according to embodiment 86, wherein the chronic urticaria is chronic inducible urticaria. 88. The use according to embodiment 87, wherein the chronically induced urticaria is cold urticaria. 89. The use according to embodiment 87, wherein the chronically induced urticaria is symptomatic dermatographia. 90. The use according to embodiment 87, wherein the chronically induced urticaria is cholinergic urticaria. 91. The use according to embodiment 86, wherein the chronic urticaria is chronic spontaneous urticaria. 92. The use of any one of embodiments 86 to 91, wherein the medicament is prepared to be administered in combination with a second therapeutic agent. 93. The use of embodiment 92, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. 94. An antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharma- ceutical acceptable carrier for use in a method for preventing, treating or managing chronic urticaria in a subject. 95. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 94, wherein human KIT comprises the amino acid sequence of SEQ ID NO:1. 96. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 94 or 95, wherein the antibody specifically binds to the D4 or D5 region of human KIT. 97. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94 to 96, wherein the antibody comprises an altered (e.g., mutated) Fc region or domain. 98. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94 to 97, wherein the antibody comprises a modified (e.g., mutated) human Fc region or domain. 99. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94 to 98, wherein the antibody has reduced Fc receptor binding activity (particularly reduced FcγR binding activity). 100. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94 to 99, wherein the antibody does not induce significant degranulation of human mast cells expressing FcgRI. 101. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94 to 100, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity. 102. The antibody, (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; The antibody, conjugate or pharmaceutical composition for use according to any one of embodiments 94 to 101, comprising: 103. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 102, wherein the chronic urticaria is chronic inducible urticaria. 104. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 103, wherein the chronic induced urticaria is cold urticaria. 105. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 103, wherein the chronic induced urticaria is symptomatic dermatographia. 106. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 103, wherein the chronically induced urticaria is cholinergic urticaria. 107. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 102, wherein the chronic urticaria is chronic spontaneous urticaria. 108. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 102 to 107, wherein the method further comprises administering to the subject a second therapeutic agent. 109. The antibody, conjugate, or pharmaceutical composition for use according to embodiment 108, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulatory agent, or an anti-inflammatory agent. [Brief description of the drawings]

[0040] (4. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1]1 shows the amino acid sequence of full-length human KIT (SEQ ID NO:1), GenBank™ Accession No. AAC50969. The first through fifth extracellular Ig-like domains (i.e., D1, D2, D3, D4, and D5) are shown; "{" indicates the amino terminal residue of each domain and "}" indicates the carboxyl terminal residue of each domain. The D1 domain is shown at P34-R112, the D2 domain is shown at D113-P206, the D3 domain is shown at A207-D309, the D4 domain is shown at K310-N410, the hinge region between D4 and D5 is located at V409-N410, and the D5 domain is shown at T411-K509. Additionally, the D1 / D2 hinge region is located at D113 to L117; the D2 / D3 hinge region is located at P206 to A210; and the D3 / D4 hinge region is located at D309 to G311. The D4 / D5 region includes K310 to K509. The transmembrane domain includes residues F525 to Q545, and the kinase domain includes residues K589 to S933.

[0041] [Diagram 2] 2A-2E show the effect of mAb1, a specific anti-KIT antibody according to the invention, on plasma tryptase levels.

[0042] [Diagram 3] 3A and 3B show the further effect of mAb1, a specific anti-KIT antibody according to the invention, on plasma tryptase levels.

[0043] [Figure 4] FIG. 4 shows the effect of a particular anti-KIT antibody according to the invention, mAb1, on plasma stem cell factor (SCF) levels.

[0044] [Diagram 5] FIG. 5 shows the effect of a specific anti-KIT antibody according to the invention, mAb1, and the corresponding antibody mAbc having the same variable region sequences but unmutated (wild-type) human IgG1 sequences, on SCF-induced activation of wild-type KIT and downstream intracellular signaling pathways.

[0045] [Figure 6] FIG. 6 shows the effect on SCF-dependent cell proliferation of a particular anti-KIT antibody according to the invention, mAb1, and the corresponding antibody mAbc having the same variable region sequences but unmutated (wild-type) human IgG1 sequences.

[0046] [Figure 7] FIG. 7 shows the binding affinity of a particular anti-KIT antibody according to the invention, mAb1, and the corresponding antibody mAbc having the same variable region sequences but unmutated (wild-type) human IgG1 sequences, to recombinant human Fc-gamma receptor (FcγR) and human fetal Fc receptor (FcRn).

[0047] [Figure 8] 8A-8N show binding curves of a specific anti-KIT antibody according to the invention, mAb1, and a corresponding antibody mAbc with the same variable region sequence but with unmutated (wild type) human IgG1 sequence to recombinant human Fc-gamma receptor (FcγR) and human fetal Fc receptor (FcRn). FIG. 8A shows the binding curve of mAb1 to FcγRI. FIG. 8B shows the binding curve of mAb1 to FcγRIIa. FIG. 8C shows the binding curve of mAb1 to FcγRIIb. FIG. 8D shows the binding curve of mAb1 to FcγRIIIa. FIG. 8E shows the binding curve of mAb1 to FcγRIIIb. FIG. 8F shows the binding curve of mAb1 to FcRn (pH 6.0). FIG. 8G shows the binding curve of mAb1 to FcRn (pH 7.2). FIG. 8H shows the binding curve of mAbc to FcγRI. Figure 8I shows the binding curve of mAbc to FcγRIIa. Figure 8J shows the binding curve of mAbc to FcγRIIb. Figure 8K shows the binding curve of mAbc to FcγRIIIa. Figure 8L shows the binding curve of mAbc to FcγRIIIb. Figure 8M shows the binding curve of mAbc to FcRn (pH 6.0). Figure 8N shows the binding curve of mAbc to FcRn (pH 7.2).

[0048] [Figure 9] FIG. 9 shows the effect on antibody-dependent cellular cytotoxicity (ADCC) activity of a particular anti-KIT antibody according to the invention, mAb1, and the corresponding antibody mAbc having the same variable region sequences but unmutated (wild-type) human IgG1 sequences.

[0049] [Figure 10] 10 shows the effect of a particular anti-KIT antibody, mAb1, according to the invention on specific cytokine production. Conditions shown in each bar graph are, from left to right: PHA, LPS, huIgG1 (soluble), mAb1 0.02 nM (soluble), mAb1 0.2 nM (soluble), mAb1 40 nM (soluble), mAb1 0.02 nM (dry coating), mAb1 0.2 nM (dry coating), and mAb1 40 nM (dry coating).

[0050] [Figure 11] FIG. 11 is a schematic diagram showing the role of KIT signaling in mast cells and the effect of mAb1 on the KIT receptor.

[0051] [Figure 12] Figures 12A-12D show that a single dose of mAb1 resulted in rapid and sustained responses with a 95% complete response (CR) rate in patients with chronically inducible urticaria (CIndU). 10 / 10 cold urticaria (ColdU) patients achieved CR (Figure 12A). 8 / 9 symptomatic dermatitis (SD) patients achieved CR and 1 / 9 SD patients achieved partial response (PR) (Figure 12B). CR = negative provocation test below 4°C or 0 pins; PR = improvement at 4°C or 2 or more pins; maximum response for each patient is shown. TempTest® results over time in ColdU patients are shown in Figure 12C. Among complete ColdU patients (n=8), CR was maintained for a median duration of 77 days (Figure 12C). FricTest® results over time in SD patients are shown in Figure 12D. Among patients with complete SD (n=6), CR was maintained for a median duration of 57 days (FIG. 12D).

[0052] [Figure 13] 13A-13B show overall disease improvement as evidenced by Physician Global Assessment (Phys-GA) and Patient Global Assessment (Pat-GA). Phys-GA and Pat-GA assess disease severity using a Likert scale of 0-3, where 0 is none and 3 is severe.

[0053] [Figure 14] Figures 14A-14D show that mAb1 treatment significantly depleted skin mast cells and serum tryptase. Figure 14A shows that mAb1 reduced skin mast cell numbers (n=14, * means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001). Figure 14B shows that mAb1 reduced serum tryptase to below the limit of detection in all patients (tryptase values ​​below the limit of assay quantification (LLoQ=1 ng / mL) were normalized to 0). Figure 14C shows mast cell and tryptase kinetics. Figure 14D shows that skin mast cell numbers correlated with serum tryptase levels (p<0.0001; R2=0.45).

[0054] [Figure 15] Figures 15A-15D show that the kinetics of skin mast cells and tryptase depletion reflected the lowering of the induction threshold. Figure 15A shows mast cell kinetics and TempTest® results over time in a ColdU patient. Figure 15B shows mast cell kinetics and FricTest® results over time in a SD patient. Figure 15C shows tryptase kinetics and TempTest® results over time in a ColdU patient (tryptase values ​​below the LLoQ were normalized to 0; a critical temperature threshold below 4°C (negative test) was set to a value of 3°C). Figure 15D shows tryptase kinetics and FricTest® results over time in a SD patient.

[0055] [Figure 16]Figures 16A-16D show that hematological parameters remained within normal ranges in most cases, with mild, transient and asymptomatic decreases in hemoglobin and white blood cell (WBC) parameters. Figure 16A shows hemoglobin (HgB) levels over time. Figure 16B shows WBC counts over time. Figure 16C shows platelet counts over time. Figure 16D shows absolute neutrophil counts (ANC) over time. In each graph, the shaded area represents the corresponding normal range.

[0056] [Figure 17] Figures 17A-17B show that a single dose of 3 mg / kg mAb1 resulted in rapid and sustained improvement in urticaria control in cold urticaria (ColdU) patients (n=10, see Figure 17A) and symptomatic dermatographia (SD) patients (n=10, see Figure 17B). Urticaria Control Test (UCT) score=16 indicates complete control of urticaria, UCT score ≥12 indicates well-controlled urticaria, and UCT score <12 indicates poorly controlled urticaria. Each graph shows the mean UCT score ± SEM.

[0057] [Figure 18] Figures 18A-18B show that a single dose of 3 mg / kg mAb1 resulted in rapid and sustained improvement in urticaria control in ColdU and SD patients. Figure 18A shows that 100% of patients achieved a "well controlled" state (UCT score ≧12) by 8 weeks. Figure 18B shows that 63% of patients achieved a "completely controlled" state (UCT score = 16) by 8 weeks.

[0058] [Figure 19] Figures 19A-19B show that mAb1 significantly reduced the impact of disease on quality of life in patients with cold urticaria (ColdU, n=10, see Figure 19A) and symptomatic dermatographia (SD, n=10, see Figure 19B). Mean DLQI scores ± SEM are shown.

[0059] [Figure 20] Figures 20A-20B show that mAb1 significantly reduced the impact of disease on quality of life in patients with cold urticaria (ColdU) and symptomatic dermatographia (SD). Figure 20A shows that 93% of patients achieved a clinically significant improvement in quality of life by 4 weeks. †: A reduction in DLQI ≥ 4 points is the minimal clinically important difference (MCID). *: Only patients with a baseline DLQI score ≥ 4 were included. Figure 20B shows that 58% of patients reported no disease affecting quality of life by 4 weeks. ‡: All responses provided at each week were included.

[0060] [Figure 21] Figures 21A-21B show that a single dose of 3 mg / kg mAb1 produced rapid and sustained improvement in provocation testing with 95% complete response (CR) and significant tryptase reduction. Figure 21A shows that mAb1 produced rapid and sustained improvement in provocation testing with 95% complete response. Disease activity was assessed by critical temperature threshold (CTT) by TempTest® for cold urticaria (ColdU) and critical friction threshold (CFT) by FricTest® for symptomatic dermographism (SD). *: Critical temperature threshold (negative test) below 4°C was taken as a value of 3°C. 10 / 10 ColdU and 9 / 10 SD patients experienced a CR during the study. CR = negative provocation test below 4°C (for ColdU) or 0 pins (for SD). Graphs show mean ± SEM. Figure 21B shows that mAb1 produced a rapid, durable and significant reduction in tryptase. Tryptase values ​​below the lower limit of quantification (1 ng / mL) were normalized to 0. Graphs show mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] (5. Detailed Description) Provided herein are antibodies that immunospecifically bind to human KIT (e.g., KIT polypeptides comprising human KIT D4 or D5 domains), and antigen-binding fragments thereof, and conjugates thereof. In a preferred embodiment, provided herein are antibodies that immunospecifically bind to human KIT, particularly antibodies with reduced Fc receptor binding activity (particularly reduced FcγR binding activity), and improved pharmacokinetics, reduced degranulation of human mast cells expressing FcgRI, and / or reduced Fc receptor-dependent KIT agonist activity. In certain embodiments, provided herein are antigen-binding fragments that immunospecifically bind to human KIT. Also provided are isolated nucleic acids (polynucleotides) encoding such antibodies or antigen-binding fragments thereof. Further provided are vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding such antibodies or antigen-binding fragments thereof. Also provided are methods of making such antibodies, cells, e.g., host cells. Also provided herein are methods and uses for preventing, treating, or managing a KIT-related disorder or disease, comprising administering an antibody, or an antigen-binding fragment thereof, or a conjugate thereof, as described herein. Also provided herein are methods for diagnosing a KIT-related disorder or disease, comprising contacting a sample with an antibody, or an antigen-binding fragment thereof, as described herein, and determining the expression level of KIT in the sample relative to a reference sample (e.g., a control sample). Also provided herein are methods and uses for inhibiting KIT activity in a cell expressing KIT, comprising contacting the cell with an effective amount of an antibody, or an antigen-binding fragment thereof, as described herein. Also provided herein are methods for inducing or enhancing cell differentiation or apoptosis in a cell expressing KIT, comprising contacting the cell with an effective amount of an antibody, or an antigen-binding fragment thereof, as described herein.

[0062] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a humanized anti-KIT antibody or antigen-binding fragment thereof provided herein) to a subject or patient (e.g., a human), e.g., by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0063] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapy (e.g., an antibody or pharmaceutical composition provided herein) that is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease and / or symptoms associated therewith. These terms also encompass the amount necessary to reduce, delay, or ameliorate the development or progression of a given disease, reduce, delay, or ameliorate the recurrence, occurrence, or onset of a given disease, and / or improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g., a therapy other than an anti-KIT antibody provided herein). In some embodiments, the term "effective amount" as used herein also refers to the amount of an antibody described herein to achieve a particular result, such as reducing the number and / or activity of mast cells, reducing the number and / or activity of eosinophils, inhibiting (e.g., partially inhibiting) KIT biological activity of cells, such as inhibiting cell proliferation or cell survival, or enhancing or inducing apoptosis or cell differentiation.

[0064] As used herein, the terms "D4 or D5 region" or "D4 / D5 domain" refer to a region within the KIT polypeptide that spans the fourth Ig-like extracellular ("D4") domain, the fifth Ig-like extracellular ("D5") domain, and the hinge region between the D4 and D5 domains (the "D4-D5 hinge region") from amino-terminus to carboxyl-terminus in the following order: D4, D4-D5 hinge region, and D5. As used herein, amino acids V308 to H515 of FIG. 1 are considered an example of a D4 / D5 region or domain.

[0065] As used herein, the terms "KIT" or "KIT receptor" or "KIT polypeptide" refer to any form of full-length KIT, including, but not limited to, native KIT, a KIT isoform, an interspecies KIT homolog, or a KIT variant, e.g., a naturally occurring variant (e.g., an allelic or splice variant, or a mutant, e.g., a somatic mutant), or an artificially constructed variant (e.g., a recombinant or chemically modified variant). KIT is a type III receptor tyrosine kinase encoded by the c-kit gene (see, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464; Yarden et al., EMBO J., 1987, 6:3341-3351; Mol et al., J. Biol. Chem., 2003, 278:31461-31464). GenBank™ Accession No. NM 000222 provides an exemplary human KIT nucleic acid sequence. GenBank™ Accession Nos. NP 001087241, PI 0721, and AAC50969 provide exemplary human KIT amino acid sequences. GenBank™ Accession No. AAH75716 provides exemplary mouse KIT amino acid sequences. Native KIT contains five extracellular immunoglobulin (Ig)-like domains (D1, D2, D3, D4, D5), a single transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (see, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464). An exemplary amino acid sequence of the D4 / D5 region of human KIT is provided in FIG. 1 from amino acid residues V308 to H515.In a specific embodiment, KIT is human KIT. In certain embodiments, KIT can exist as a monomer, dimer, multimer, native form, or denatured form.

[0066] As used herein, the term "in combination with" in the context of the administration of other therapies refers to the use of more than one therapies. The use of the term "in combination" does not restrict the order in which the therapies are administered. Therapies may be administered, for example, sequentially, sequentially, in parallel, or simultaneously.

[0067] As used herein, the term "KIT-associated disorder" or "KIT-associated disease" is used interchangeably and refers to any disease caused, associated with, or resulting from, in whole or in part, KIT expression and / or activity or lack thereof. In one aspect, the KIT-associated disorder or disease may be known to, or may be identified by, a person skilled in the art. In certain embodiments, the KIT-associated disease or disorder is associated with KIT expression and / or activity. For example, KIT expression and / or activity may be combined with one or more other factors (e.g., mutation, or expression and / or activity of another gene) to contribute to the development and / or progression of the KIT-associated disease or disorder. In certain embodiments, the KIT-associated disease or disorder is associated with one or more mutations of KIT.

[0068] In some embodiments, the KIT-related disorder is mast cell-related disorder, eosinophil-related disorder, cancer, asthma, inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, gastrointestinal disorder, or fibrosis.In some embodiments, the KIT-related disorder is fibrosis or inflammatory disorder, for example, inflammatory bowel disease (IBD), for example, Crohn's disease (CD) or ulcerative colitis (UC).In other embodiments, the KIT-related disorder is cancer, for example, lung cancer (for example, small cell lung cancer), leukemia, neuroblastoma, melanoma, sarcoma (for example, Ewing's sarcoma), or gastrointestinal stromal tumor (GIST).In a specific embodiment, the KIT-related disorder is a mast cell-related disorder.In a specific embodiment, the KIT-related disorder is an eosinophil-related disorder, for example, eosinophilic esophagitis (EoE).

[0069] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a KIT-related disease (e.g., cancer, inflammatory disorder, or fibrosis) resulting from the administration of one or more therapies (including but not limited to, the administration of one or more prophylactic or therapeutic agents, e.g., an antibody provided herein).

[0070] As used herein, the terms "manage," "managing," and "management" refer to the non-cure beneficial effect that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent). In certain embodiments, a subject is administered one or more therapies (e.g., a prophylactic or therapeutic agent, e.g., an antibody described herein) to "manage" a disorder, or one or more symptoms thereof, to prevent progression or worsening of the disorder.

[0071] As used herein, the terms "prevent," "impede," or "impeding" in the context of a disorder refer to complete or partial inhibition (e.g., less than 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or prevention of the onset, recurrence, development, or spread of a disorder and / or symptoms associated therewith, brought about by administration of a therapy or combination of therapies provided herein (e.g., a prophylactic or therapeutic agent, e.g., a combination of antibodies described herein).

[0072] As used herein, the term "prophylactic agent" refers to any agent that can completely or partially inhibit the occurrence, recurrence, development or spread of a disorder and / or symptoms associated therewith in a subject. In certain embodiments, the term "prophylactic agent" refers to an antibody described herein. In certain other embodiments, the term "prophylactic agent" refers to an agent other than an antibody described herein. Typically, a prophylactic agent is an agent that is known to be useful, has been used, or is currently used to prevent a disorder and / or symptoms associated therewith, or to hinder the onset, development, progression, and / or severity of a disorder and / or symptoms associated therewith. In a specific embodiment, the prophylactic agent is a human anti-KIT antibody, e.g., a humanized or fully human anti-KIT monoclonal antibody.

[0073] As used herein, the term "side effects" or "adverse effects" encompasses undesirable and harmful effects of a therapy (e.g., a prophylactic or therapeutic agent). Undesirable effects are not necessarily harmful. Adverse effects from a therapy (e.g., a prophylactic or therapeutic agent) can be harmful or unpleasant or dangerous. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, loss of appetite, abdominal cramps, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, xerostomia, and loss of appetite, rash or swelling at the site of administration, flu-like symptoms such as fever, chills and fatigue, gastrointestinal upset, and allergic reactions. Additional undesirable effects experienced by patients are numerous and known in the art. Many are described in the Physician's Desk Reference (71st Edition, 2017).

[0074] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkeys and humans), such as a human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a disorder. In another embodiment, the subject is a mammal, such as a human, at risk of developing a KIT-associated disorder. In another embodiment, the subject is a non-human primate. In a specific embodiment, the subject is an adult. In a specific embodiment, the subject is an adult subject at least 18 years old. In a specific embodiment, the subject is a human child. In a specific embodiment, the subject is a human child between 1 and 18 years old. In a specific embodiment, the subject is a human between 1 and 3 years old. In a specific embodiment, the subject is a human between 3 and 12 years old or between 12 and 18 years old.

[0075] As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s), composition, formulation, and / or agent(s) that can be used in the prevention, prophylaxis, treatment, management, or amelioration of a condition or disorder or one or more symptoms thereof or one or more symptoms or conditions associated therewith. In certain embodiments, the terms "therapies" and "therapy" refer to drug therapy, adjunctive therapy, radiation, surgery, biological therapy, supportive therapy, and / or other therapies useful in the prevention, treatment, management, prevention, or amelioration of a condition or disorder or one or more symptoms thereof or one or more symptoms or conditions associated therewith. In certain embodiments, the term "therapy" refers to a therapy other than an anti-KIT antibody or pharmaceutical composition thereof described herein. In a specific embodiment, "additional therapy" and "additional therapies" refer to a therapy other than a treatment using an anti-KIT antibody or pharmaceutical composition thereof described herein. In a specific embodiment, the therapy includes the use of an anti-KIT antibody described herein as an adjunctive therapy. For example, the anti-KIT antibodies described herein are used in combination with drug therapy, biological therapy, surgery, and / or supportive therapy.

[0076] As used herein, the term "therapeutic agent" refers to any agent that can be used in the prevention, treatment, management, or amelioration of a disorder and / or symptoms associated therewith. In certain embodiments, the term "therapeutic agent" refers to an anti-KIT antibody or antigen-binding fragment thereof described herein. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody described herein. In specific embodiments, a therapeutic agent is an agent that is known to be useful, has been used, or is currently used to prevent, treat, manage, or ameliorate a disorder or one or more symptoms associated therewith.

[0077] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more," and "at least one," can be used interchangeably herein.

[0078] Wherever an embodiment is described herein with the word "comprising" or otherwise, it will be understood that similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0079] As used herein, unless otherwise indicated, the term "about" or "approximately" is to be interpreted to allow for normal variations as determined by one of ordinary skill in the art, such as, for example, variations within 20%, or 10%, or 5%. In specific embodiments, the term "about" or "approximately" includes the exact value recited.

[0080] (5.1 Antibodies) Provided herein are antibodies (e.g., anti-KIT antibodies) that specifically bind to a KIT receptor (e.g., the extracellular domain of the human KIT receptor, e.g., as set forth in SEQ ID NO:1 or FIG. 1), or antigen-binding fragments thereof.

[0081] As used herein, the terms "antibody" and "immunoglobulin" and "Ig" are terms of art and may be used interchangeably herein and refer to a molecule that has an antigen binding site that immunospecifically binds to an antigen.

[0082] Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, and chimeric antibodies. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. The antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or classes (e.g., human IgG1 or IgG4) or subclasses thereof.

[0083] As used herein, an "antigen" is a moiety or molecule that contains an epitope and is therefore also specifically bound by an antibody. In a specific embodiment, the antigen that the antibody described herein binds to is KIT (e.g., human KIT), or a fragment thereof, such as the extracellular domain of KIT (e.g., human KIT) or the D4 region of KIT (e.g., human KIT).

[0084] As used herein, the terms "antigen-binding domain", "antigen-binding region", "antigen-binding fragment", and similar terms refer to the portion of an antibody molecule (e.g., complementarity determining region (CDR)) that contains amino acid residues that interact with an antigen and confer to the antibody molecule its specificity for the antigen. The antigen-binding region may be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans. The CDRs of an antibody molecule may be determined by any method known to those skilled in the art. In particular, the CDRs may be determined according to the Kabat numbering system (see Kabat et al. (1991), Sequences of Proteins of Immunological Interest (US Department of Health and Human Services, Washington, DC), 5th Edition).In certain embodiments, the CDRs of the antibody may be numbered according to: (i) the Chothia numbering scheme, referred to herein as "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; and U.S. Patent No. 7,709,226); (ii) the IMGT numbering system, as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7:132-136, and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212; (iii) the IMGT numbering system, as described, for example, in MacCallum et al., 1996, J. Mol. Biol., 262:732-745, and Martin, (iv) the Contact numbering system, which is based on the analysis of available complex crystal structures (bioinf.org.uk / abs) (see, e.g., MacCallum et al., (1996) J Mol Biol 5:732-745)). In a preferred embodiment, the antigen-binding fragment described herein comprises a full-length heavy chain Fc region or domain (e.g., a full-length human IgG1, full-length human IgG2, full-length human IgG3, or full-length human IgG4 Fc region or domain), or a partial heavy chain Fc region or domain (e.g., a partial human IgG1, partial human IgG2, partial human IgG3, or partial human IgG4 Fc region or domain).

[0085] As used herein, the term "constant region" or "constant domain" refers to those portions of antibodies, e.g., the carboxyl-terminal portions of the light and / or heavy chains, that are not directly involved in binding the antibody to an antigen, but which exhibit or contribute to various effector functions, such as interaction with Fc receptors. These terms refer to those portions of immunoglobulin molecules that have a generally more conserved amino acid sequence than the immunoglobulin variable domains.

[0086] As used herein, "epitope" is a term of art and refers to a localized region of an antigen to which an antibody can specifically bind. A region or polypeptide contributing to an epitope may be contiguous amino acids of a polypeptide, or an epitope may be a collection of two or more non-contiguous regions of a polypeptide.

[0087] As used herein, the term "heavy chain" when used in reference to an antibody refers to an antibody of the IgG subclass, e.g., IgG i , IgG 2 , IgG 3 and IgG 4 and refers to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively. In a specific embodiment, the heavy chain is a human heavy chain.

[0088] As used herein, the terms "immunospecifically bind", "immunospecifically recognize", "specifically bind", and "specifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, Biacore™, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, a molecule that immunospecifically binds to an antigen has a K a K at least 2 log, 2.5 log, 3 log, 4 log or more greater than a In another specific embodiment, a molecule that immunospecifically binds to an antigen does not cross-react with other proteins. In another specific embodiment, a molecule that immunospecifically binds to an antigen does not cross-react with other non-KIT proteins.

[0089] As used herein, an "isolated" or "purified" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is obtained, or is substantially free of chemical precursors or other chemicals from which it is chemically synthesized. In specific embodiments, the antibodies or antigen-binding fragments described herein are isolated.

[0090] The term "Kabat numbering" and similar terms are art-recognized and refer to a system for numbering the amino acid residues of the heavy and light chain variable regions of an antibody, or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391, and Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31-35 ("CDR1"), amino acid positions 50-65 ("CDR2"), and amino acid positions 95-102 ("CDR3"). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3).

[0091] As used herein, the term "light chain" when used in reference to an antibody refers to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0092] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a homogeneous or substantially homogeneous population of antibodies, with each monoclonal antibody usually recognizing a single epitope on an antigen. The term "monoclonal" is not limited to any particular antibody production method. In general, a population of monoclonal antibodies can be produced by a cell, a population of cells, or a cell line. In a specific embodiment, a "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell (e.g., a host cell producing a recombinant antibody), where the antibody immunospecifically binds to a KIT epitope (e.g., an epitope of D4 of human KIT) as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the examples provided herein. The monoclonal antibodies described herein can be produced, for example, by hybridoma methods described in Kohler et al.; Nature, 256:495 (1975), or can be isolated from a phage library, for example, using the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology (2002), 5th ed., Ausubel et al., eds., John Wiley and Sons, New York:). In specific embodiments, monoclonal antibodies are monospecific antibodies in that their antigen-binding regions are specific for the same epitope. In further specific embodiments, monoclonal monospecific antibodies can be monovalent (having one antigen-binding region) or multivalent (having two or more antigen-binding regions), e.g., bivalent (having two antigen-binding regions).

[0093] As used herein, the term "naked antibody" refers to an antibody that is not linked, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In a specific embodiment, a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery, e.g., glycosylation enzymes, of the host cell. In certain embodiments, a naked antibody is not glycosylated when it is expressed by a host cell that does not have its own glycosylation machinery, e.g., glycosylation enzymes. In certain embodiments, a naked antibody is a complete antibody, and in other embodiments, a naked antibody is an antigen-binding fragment of a complete antibody, such as a Fab antibody.

[0094] As used herein, the term "polyclonal antibody" refers to an antibody population that includes a variety of different antibodies directed against the same epitope and against different epitopes within an antigen or antigens. Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology (2002), 5th ed., Ausubel et al., eds., John Wiley and Sons, New York:).

[0095] As used herein, the term "recombinant human antibody" includes human antibodies that are isolated, prepared, expressed, or produced by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., mouse, rabbit, goat, or cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means including, for example, synthesis, genetic modification of DNA sequences encoding human immunoglobulin sequences, or production by splicing of human immunoglobulin-encoding sequences, e.g., human immunoglobulin gene sequences, to other such sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, the amino acid sequences of such recombinant human antibodies are so modified that the amino acid sequences of the VH and / or VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that do not naturally occur in the human antibody germline repertoire in vivo. As a non-limiting example, a recombinant human antibody can be obtained by assembling several human sequence fragments into the composite human sequence of a recombinant human antibody.

[0096] As used herein, the term "variable region" or "variable domain" refers to that portion of an antibody, generally a light or heavy chain portion, usually about the amino-terminal 110-120 amino acids in mature heavy chains and about 90-100 amino acids in mature light chains, which differs widely in sequence among antibodies and is used with respect to the binding and specificity of a particular antibody for its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in variable domains are called framework regions (FRs).

[0097] Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily involved in the interaction of the antibody with the antigen. In a specific embodiment, the numbering of the amino acid positions of the antibodies described herein is according to the EU index, as found in Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 ("Kabat et al.").In certain embodiments, the CDRs of the antibody may be numbered according to: (i) the Chothia numbering scheme, referred to herein as "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; and U.S. Patent No. 7,709,226); (ii) the IMGT numbering system, as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7:132-136, and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212; (iii) the IMGT numbering system, as described, for example, in MacCallum et al., 1996, J. Mol. Biol., 262:732-745, and Martin, The AbM numbering system described in A., Antibody Engineering, Protein Sequence and Structure Analysis of Antibody Variable Domains, Kontermann and Dubel (eds.), Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001); or (iv) the Contact numbering system based on the analysis of available complex crystal structures (bioinf.org.uk / abs) (see, e.g., MacCallum et al., (1996) J Mol Biol 5:732-745). In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FR).As a non-limiting example, the variable regions described herein are obtained by assembling two or more fragments of human sequences into a composite human sequence.

[0098] In a specific embodiment, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a light chain variable region ("VL") comprising VL CDR1-3, and a heavy chain variable region ("VH") comprising VH CDR1-3, as set forth in Table 1. In a specific embodiment, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a light chain variable region ("VL") comprising VL CDR1-3, and a heavy chain variable region ("VH") comprising VH CDR1-3, as set forth in Table 2 (Set 1 or Set 2). In a specific embodiment, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a light chain variable region ("VL") comprising VL CDR1-3, and a heavy chain variable region ("VH") comprising VH CDR1-3, as set forth in Table 3 (AbM CDRs or contact CDRs).

[0099] In a specific embodiment, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a VL comprising VL CDR1-3 (SEQ ID NOs:2-4) set forth in Table 1, and a VH comprising VH CDR1-3 (SEQ ID NOs:5-7) set forth in Table 1. In certain embodiments, such an anti-KIT antibody is a naked antibody. In specific embodiments, such an anti-KIT antibody is a bivalent monospecific antibody. In specific embodiments, such an anti-KIT antibody is a bispecific antibody. In certain embodiments, such an anti-KIT antibody is not a bispecific antibody.

[0100] Table 1: CDR amino acid sequences [Table 1]

[0101] Table 2: CDR amino acid sequences [Table 2]

[0102] Table 3: CDR amino acid sequences [Table 3]

[0103] In certain embodiments, the anti-KIT antibodies (e.g., humanized antibodies) provided herein are (i) Amino acid sequence: [ka] (where X K1 ~X K6 is any amino acid; and (ii) Amino acid sequence: [ka] (where X H1 ~X H8 is any amino acid) Includes.

[0104] In certain embodiments, X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid having an aliphatic or aliphatic hydroxyl side chain, and X K3 is an amino acid having an aliphatic hydroxyl side chain, and X K4 is an amino acid having an aliphatic hydroxyl side chain or is P; X K5 is an amino acid having a charged or acidic side chain, and X K6 is an amino acid with an aromatic side chain, and X H1 is an amino acid having an aliphatic side chain, and X H2 is an amino acid having an aliphatic side chain, and X H3 is an amino acid with a polar or basic side chain, and X H4 is an amino acid having an aliphatic side chain, and X H5 is an amino acid having an aliphatic side chain, and X H6 is an amino acid with an acidic side chain, and X H7is an amino acid having an acidic or amide derivative side chain, and X H8 is an amino acid having an aliphatic hydroxyl side chain.

[0105] In a specific embodiment, X K1 is the amino acid F or S, and X K2 is the amino acid A or S, and X K3 is the amino acid T or S, and X K4 is the amino acid S or P, and X K5 is the amino acid D or T, and X K6 is an amino acid F or Y, and X H1 is an amino acid L or V, and X H2 is an amino acid L or V, and X H3 is the amino acid K or R, and X H4 is an amino acid V or A, and X H5 is an amino acid L or I, and X H6 is the amino acid E or D, and X H7 is an amino acid Q or E, and X H8 is the amino acid S or T.

[0106] In certain embodiments, the anti-KIT antibodies (e.g., humanized antibodies) provided herein are (i) Amino acid sequence: [ka] (where X K1 ~X K6 is any amino acid; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; Includes.

[0107] In certain embodiments, X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid having an aliphatic or aliphatic hydroxyl side chain, and X K3is an amino acid having an aliphatic hydroxyl side chain, and X K4 is an amino acid having an aliphatic hydroxyl side chain or is P; X K5 is an amino acid having a charged or acidic side chain, and X K6 is an amino acid with an aromatic side chain.

[0108] In a specific embodiment, X K1 is the amino acid F or S, and X K2 is the amino acid A or S, and X K3 is the amino acid T or S, and X K4 is the amino acid S or P, and X K5 is the amino acid D or T, and X K6 is the amino acid F or Y.

[0109] In certain embodiments, the anti-KIT antibodies (e.g., humanized antibodies) provided herein are (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) Amino acid sequence: [ka] (where X H1 ~X H8 is any amino acid) Includes.

[0110] In certain embodiments, X H1 is an amino acid having an aliphatic side chain, and X H2 is an amino acid having an aliphatic side chain, and X H3 is an amino acid with a polar or basic side chain, and X H4 is an amino acid having an aliphatic side chain, and X H5 is an amino acid having an aliphatic side chain, and X H6 is an amino acid with an acidic side chain, and X H7 is an amino acid having an acidic or amide derivative side chain, and X H8is an amino acid having an aliphatic hydroxyl side chain.

[0111] In a specific embodiment, X H1 is an amino acid L or V, and X H2 is an amino acid L or V, and X H3 is the amino acid K or R, and X H4 is an amino acid V or A, and X H5 is an amino acid L or I, and X H6 is the amino acid E or D, and X H7 is an amino acid Q or E, and X H8 is the amino acid S or T.

[0112] In specific aspects, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a heavy chain variable region ("VH") comprising an amino acid sequence selected from Table 4 (SEQ ID NOs:8-12) and / or a light chain variable region ("VL") comprising an amino acid sequence selected from Table 5 (SEQ ID NOs:13-16). In certain embodiments, such an anti-KIT antibody is a naked antibody. In specific embodiments, such an anti-KIT antibody is a bivalent monospecific antibody. In specific embodiments, such an anti-KIT antibody is a bispecific antibody. In certain embodiments, such an anti-KIT antibody is not a bispecific antibody.

[0113] Table 4: VH amino acid sequences [Table 4]

[0114] Table 5: VL amino acid sequences [Table 5]

[0115] In specific aspects, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 8, and / or a VL comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 8, and / or a VL comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 8, and / or a VL comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 8, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0116] In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:9, and / or a VL comprising the amino acid sequence of SEQ ID NO:13. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:9, and / or a VL comprising the amino acid sequence of SEQ ID NO:14. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:9, and / or a VL comprising the amino acid sequence of SEQ ID NO:15. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:9, and / or a VL comprising the amino acid sequence of SEQ ID NO:16.

[0117] In one embodiment, the anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 10, and / or a VL comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, the anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 10, and / or a VL comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 10, and a VL comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 10, and / or ...5. In one embodiment, the anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 10, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0118] In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, and / or a VL comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, and / or a VL comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, and / or a VL comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0119] In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 12, and / or a VL comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 12, and / or a VL comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 12, and / or a VL comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, an anti-KIT antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 12, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16.

[0120] In a specific embodiment, the anti-KIT antibodies (e.g., humanized antibodies) provided herein are (i) a VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, at least 88% identical to SEQ ID NO: 14, at least 87% identical to SEQ ID NO: 15, or at least 84% identical to SEQ ID NO: 16; and (ii) a VH comprising an amino acid sequence that is at least 93% identical to SEQ ID NO:8, at least 92% identical to SEQ ID NO:9, at least 90% identical to SEQ ID NO:10, at least 87% identical to SEQ ID NO:11, or at least 86% identical to SEQ ID NO:12. Includes.

[0121] Previous anti-KIT antibodies have been found to induce degranulation of FcgRI-expressing human mast cells and / or exhibit Fc receptor-dependent KIT agonist activity, which can result in undesirable infusion-related reactions (IRR) among other side effects.

[0122] In various embodiments, the anti-KIT antibodies or antigen-binding fragments described herein comprise an altered (e.g., mutated) Fc region or domain (e.g., an altered (e.g., mutated) human IgG Fc region or domain, e.g., an altered (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain). Preferably, the anti-KIT antibodies or antigen-binding fragments described herein have reduced Fc receptor binding activity (particularly reduced FcγR binding activity), do not induce degranulation of FcgRI-expressing human mast cells, and / or do not exhibit Fc receptor-dependent KIT agonist activity. In certain embodiments, one or more of these properties of the anti-KIT antibodies or antigen-binding fragments result from the altered (e.g., mutated) Fc region or domain.

[0123] In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein has reduced Fc receptor binding activity (particularly reduced FcγR binding activity). In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein does not have significant Fc receptor (particularly FcγR) binding activity. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein has no detectable Fc receptor (particularly FcγR) binding activity. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein has at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less Fc receptor (particularly FcγR) binding activity compared to a suitable control antibody or antigen-binding fragment. Where an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain, in a preferred embodiment, a suitable control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL but with a wild-type (unmodified) Fc region or domain of the same isotype. In a particular embodiment, an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) human IgG1 Fc region or domain and has the same VH and VL but with a wild-type (unmodified) human IgG1 The Fc receptor (particularly FcγR) binding activity is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less than a corresponding antibody or antigen-binding fragment having an Fc region or domain.

[0124] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not induce significant degranulation of human mast cells expressing FcgRI (e.g., as determined by the percentage release of beta-hexosaminidase from human mast cells in culture (e.g., in the presence of IFN-gamma)). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not induce detectable degranulation of human mast cells expressing FcgRI (e.g., as determined by the percentage release of beta-hexosaminidase from human mast cells in culture (e.g., in the presence of IFN-gamma)). In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein induce degranulation of human mast cells expressing FcgRI (e.g., as determined by the percent release of beta-hexosaminidase from human mast cells in culture (e.g., in the presence of IFN-gamma)) by at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less compared to a suitable control antibody or antigen-binding fragment. In certain embodiments, the release of beta-hexosaminidase from human mast cells in culture in the presence of IFN-gamma is reduced by more than 50% by the anti-KIT antibodies or antigen-binding fragments described herein compared to a suitable control antibody or antigen-binding fragment. In certain embodiments, the release of beta-hexosaminidase from human mast cells in culture in the presence of IFN gamma is reduced by more than 60%, more than 70%, or more than 80% by an anti-KIT antibody or antigen-binding fragment described herein compared to a suitable control antibody or antigen-binding fragment.Where an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain), in a preferred embodiment, a suitable control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL, but with a wild-type (unmodified) Fc region or domain of the same isotype. In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) human IgG1 Fc region or domain and induces at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less degranulation of human mast cells expressing FcgRI (e.g., as determined by the % release of beta-hexosaminidase from human mast cells in culture (e.g., in the presence of IFN-gamma)) compared to a corresponding antibody or antigen-binding fragment having an identical VH and VL but a wild-type (unmodified) human IgG1 Fc region or domain. In certain embodiments, the release of beta-hexosaminidase from human mast cells during culture in the presence of IFN gamma is reduced by more than 50% by an anti-KIT antibody or antigen-binding fragment described herein that comprises a modified (e.g., mutated) human IgG1 Fc region or domain compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but with a wild-type (unmodified) human IgG1 Fc region or domain. In certain embodiments, the release of beta-hexosaminidase from human mast cells during culture in the presence of IFN gamma is reduced by more than 60%, more than 70%, or more than 80% by an anti-KIT antibody or antigen-binding fragment described herein that comprises a modified (e.g., mutated) human IgG1 Fc region or domain compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but with a wild-type (unmodified) human IgG1 Fc region or domain.

[0125] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not exhibit significant Fc receptor-dependent KIT agonist activity (e.g., as determined by KIT phosphorylation). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not exhibit detectable Fc receptor-dependent KIT agonist activity (e.g., as determined by KIT phosphorylation). In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein induce Fc receptor-dependent KIT activity at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less (e.g., as determined by KIT phosphorylation) compared to a suitable control antibody or antigen-binding fragment. In certain embodiments, Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation by cross-linked Fc receptors) is reduced by more than 50% by the anti-KIT antibody or antigen-binding fragment described herein compared to a suitable control antibody or antigen-binding fragment. In certain embodiments, Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation by cross-linked Fc receptors) is reduced by more than 60%, more than 70%, or more than 80% by the anti-KIT antibody or antigen-binding fragment described herein compared to a suitable control antibody or antigen-binding fragment. When the anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain), in a preferred embodiment, a suitable control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL but a wild-type (unmodified) Fc region or domain of the same isotype.In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein comprise a modified (e.g., mutated) human IgG1 Fc region or domain and induce at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less Fc receptor-dependent KIT activity (e.g., as determined by KIT phosphorylation) compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but a wild-type (unmodified) human IgG1 Fc region or domain. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein, even when cross-linked on THP-1 cells, do not exhibit significant or detectable Fc receptor-dependent KIT agonist activity as described herein. In certain embodiments, Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation by cross-linked Fc receptor) is reduced by more than 50% with an anti-KIT antibody or antigen-binding fragment described herein that comprises a modified (e.g., mutated) human IgG1 Fc region or domain compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but with a wild-type (unmodified) human IgG1 Fc region or domain. In certain embodiments, Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation by cross-linked Fc receptor) is reduced by more than 60%, more than 70%, or more than 80% with an anti-KIT antibody or antigen-binding fragment described herein that comprises a modified (e.g., mutated) human IgG1 Fc region or domain compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but with a wild-type (unmodified) human IgG1 Fc region or domain.

[0126] In various embodiments, the anti-KIT antibodies or antigen-binding fragments described herein (1) reduce disease activity in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (2) decrease skin mast cell numbers in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (3) decrease tryptase levels in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (4) decrease tryptase levels in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), or (5) decrease disease activity in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment). (5) improve quality of life in CIndU patients (e.g., CIndU patients whose CIndU are refractory to antihistamine treatment); and / or (6) maintain hematological parameters (e.g., hemoglobin (HgB) levels, white blood cell (WBC) count, platelet count, and / or absolute neutrophil count (ANC)) within the normal range in patients such as CIndU patients (e.g., CIndU patients whose CIndU are refractory to antihistamine treatment).

[0127] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly reduce the critical temperature threshold in a TempTest® for a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) compared to the pre-treatment value. In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments described herein can reduce the critical temperature threshold in a TempTest® for a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, or at least 20°C (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) compared to the pre-treatment value. In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0128] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly reduce the number of pins in the FricTest® for CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) compared to the number of pins before treatment. In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments described herein can reduce the number of pins in the FricTest® for CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) by at least 1, at least 2, at least 3, or at least 4 (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibodies or antigen-binding fragments) compared to the number of pins before treatment. In a specific embodiment, the effect of the anti-KIT antibodies or antigen-binding fragments lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0129] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly improve Physician Global Assessment (Phys-GA) and / or Patient Global Assessment (Pat-GA) compared to pre-treatment levels. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can improve Physician Global Assessment (Phys-GA) and / or Patient Global Assessment (Pat-GA) by lowering a Likert scale (from 0 to 3, where 0 is none and 3 is severe) by at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, or at least 1.3 (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) compared to pre-treatment levels. In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0130] In certain embodiments, the anti-KIT antibody or antigen-binding fragment described herein can significantly reduce the number of skin mast cells in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) compared to the number before treatment. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein can reduce the number of skin mast cells in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by at least 20%, at least 40%, at least 60%, or at least 80% (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) compared to the number before treatment. In a specific embodiment, the effect of the anti-KIT antibody or antigen-binding fragment lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0131] In certain embodiments, the anti-KIT antibody or antigen-binding fragment described herein can significantly reduce serum tryptase in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) compared to pre-treatment levels. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein can reduce serum tryptase in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) by at least 50%, at least 70%, or at least 90% (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) compared to pre-treatment levels. In a specific embodiment, the effect of the anti-KIT antibody or antigen-binding fragment lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0132] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly improve urticaria control in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) compared to pre-treatment levels. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment described herein can improve urticaria control in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by increasing the urticaria control test (UCT) score by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 compared to pre-treatment levels, or by increasing the UCT score by at least 12, at least 13, at least 14, at least 15, or 16 (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In a specific embodiment, the effect of the anti-KIT antibody or antigen-binding fragment lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0133] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly improve quality of life in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) compared to pre-treatment levels. In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments described herein can improve quality of life in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) by reducing the Dermatological Life Quality Index (DLQI) by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or 25, or by reducing the DLQI by up to 5, up to 4, up to 3, up to 2, up to 1, or 0 (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) compared to pre-treatment levels. In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment lasts for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0134] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein maintain hematological parameters (e.g., hemoglobin (HgB) levels, white blood cell (WBC) count, platelet count, and / or absolute neutrophil count (ANC)) in a patient within the normal range. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein maintain hematological parameters (e.g., hemoglobin (HgB) levels, white blood cell (WBC) count, platelet count, and / or absolute neutrophil count (ANC)) in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) within the normal range. In specific embodiments, the hematological parameters persist for at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks.

[0135] In various embodiments, the anti-KIT antibodies described herein have one or more of the properties described herein. In various embodiments, the antigen-binding fragments of the anti-KIT antibodies described herein have one or more of the properties described herein.

[0136] In specific embodiments, the antibodies described herein comprise an altered Fc region or domain, wherein the Fc region or domain comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue.

[0137] In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and is numbered according to the EU index as set forth in the Kabat reference: 234A, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234E, 234F, 234G, 234H, 234I, 234H, 234I, 234J, 234K ... F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D , 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R. 243W, 243L243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 26 4L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T , 2661, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 2 96H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 322Q , 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 3 28Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F , 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A, or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue selected from the group consisting of: Optionally, the Fc region or domain may include additional and / or alternative non-naturally occurring amino acid residues known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752 and WO(See US Pat. No. 5,399,136 and US Pat. No. 5,399,137, for more details.) In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, which is a human IgG2 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, which is a human IgG3 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5 or 6) amino acid modification (e.g., substitution, deletion or addition) or at least one (e.g., 1, 2, 3, 4, 5 or 6) non-naturally occurring amino acid residue that is equivalent to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, which is a human IgG4 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5 or 6) amino acid modification (e.g., substitution, deletion or addition) or at least one (e.g., 1, 2, 3, 4, 5 or 6) non-naturally occurring amino acid residue that is equivalent to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art.

[0138] In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and is numbered according to the EU index as set forth in the Kabat reference: 234A, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234E, 234F, 234G, 234H, 234I, 234H, 234I, 234J, 234K ... F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D , 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R. 243W, 243L243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 26 4L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T , 2661, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 2 96H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 322Q , 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 3 28Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F , 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A, or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue selected from the group consisting of: Optionally, the Fc region or domain may include additional and / or alternative non-naturally occurring amino acid residues known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752 and WO(See US Pat. No. 5,399,136 and US Pat. No. 5,399,137, for more details.) In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, which is a human IgG2 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, which is a human IgG3 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5 or 6) amino acid modification (e.g., substitution, deletion or addition) or at least one (e.g., 1, 2, 3, 4, 5 or 6) non-naturally occurring amino acid residue that is equivalent to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise an altered Fc region or domain, which is a human IgG4 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5 or 6) amino acid modification (e.g., substitution, deletion or addition) or at least one (e.g., 1, 2, 3, 4, 5 or 6) non-naturally occurring amino acid residue that is equivalent to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art.

[0139] In certain embodiments, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and comprises at least one unnatural amino acid at one or more positions selected from the group consisting of 239, 330, and 332, as numbered by the EU index in Kabat. In specific embodiments, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and comprises at least one unnatural amino acid at one or more positions selected from the group consisting of 239D, 330L, and 332E, as numbered by the EU index in Kabat. Optionally, the Fc region or domain can further comprise an additional unnatural amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index in Kabat. In a specific embodiment, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and comprises at least one unnatural amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat, and at least one unnatural amino acid at one or more positions is selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat. In a specific embodiment, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is a human IgG2, IgG3, or IgG4 Fc region or domain and comprises at least one unnatural amino acid residue that is equivalent(s) to the amino acid residue(s) described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art.In a specific embodiment, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is a human IgG2, IgG3, or IgG4 Fc region or domain, and comprises at least one unnatural amino acid residue at one or more positions equivalent(s) to the positions described herein for a human IgG1 Fc region or domain, as can be determined by one skilled in the art. In one embodiment, an Fc region or domain comprising such a sequence exhibits one or more Fc activities, such as binding affinity to an Fc receptor, or effector function, such as ADCC or CDC. In a specific embodiment, an Fc region or domain comprising such a sequence exhibits reduced Fc activity, such as reduced binding affinity to an Fc receptor or reduced effector function, such as ADCC or CDC. In a particular embodiment, an Fc region or domain comprising such a sequence exhibits enhanced FcRn activity, such as extended half-life.

[0140] Further non-limiting examples of Fc region or domain modifications can be found in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984; Jefferis et al., 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; Nos. 6,821,505; 6,277,375; 8,163,882; 7,355,008; 7,960,512; 8,039,592; 8,039,359; 8,101,720; 7,214,775; 7,682,610; 7,741,442; U.S. Patent Publication 2004 / 0002587 and PCT Publications WO 94 / 29351; WO 99 / 58572; WO 00 / 42072; WO 04 / 029207; WO 04 / 099249; WO 04 / 063351.

[0141] In specific embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG1 Fc region or domain comprising the non-natural amino acids 234A, 235Q, and 322Q, as numbered by the EU index in Kabat. In certain embodiments, the modified (e.g., mutated) human IgG1 Fc region or domain further comprises the non-natural amino acids 252Y, 254T, and 256E, as numbered by the EU index in Kabat.

[0142] In certain embodiments, the antibodies described herein comprise modified (e.g., mutated) human IgG2 Fc regions or domains that comprise unnatural amino acids equivalent to 234A, 235Q, and 322Q, as numbered by the EU index set forth in Kabat for human IgG1 Fc regions or domains, as can be determined by one of skill in the art. In certain embodiments, the antibodies described herein comprise modified (e.g., mutated) human IgG2 Fc regions or domains that comprise unnatural amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index set forth in Kabat for human IgG1 Fc regions or domains, as can be determined by one of skill in the art.

[0143] In certain embodiments, the antibodies described herein comprise modified (e.g., mutated) human IgG3 Fc regions or domains that comprise unnatural amino acids equivalent to 234A, 235Q, and 322Q, as numbered by the EU index set forth in Kabat for human IgG1 Fc regions or domains, as can be determined by one of skill in the art. In certain embodiments, the antibodies described herein comprise modified (e.g., mutated) human IgG3 Fc regions or domains that comprise unnatural amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index set forth in Kabat for human IgG1 Fc regions or domains, as can be determined by one of skill in the art.

[0144] In certain embodiments, the antibodies described herein comprise modified (e.g., mutated) human IgG4 Fc regions or domains that comprise unnatural amino acids equivalent to 234A, 235Q, and 322Q, as numbered by the EU index set forth in Kabat for human IgG1 Fc regions or domains, as can be determined by one of skill in the art. In certain embodiments, the antibodies described herein comprise modified (e.g., mutated) human IgG4 Fc regions or domains that comprise unnatural amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index set forth in Kabat for human IgG1 Fc regions or domains, as can be determined by one of skill in the art.

[0145] In a specific embodiment, the antibodies described herein comprise the VL and VH CDR sequences set forth in Table 1, and a modified (e.g., mutated) human IgG1 Fc region or domain that contains the unnatural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference.

[0146] In a preferred embodiment, the antibodies described herein comprise the VL and VH CDR sequences set forth in Table 1, and a modified (e.g. mutated) human IgG1 Fc region or domain comprising the unnatural amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

[0147] Thus, in one aspect, provided herein is an antibody that immunospecifically binds to human KIT, (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising the unnatural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference. The antibody comprises:

[0148] Accordingly, in a further aspect, provided herein is an antibody that immunospecifically binds to human KIT, (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising the unnatural amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index as set forth in the Kabat reference. The antibody comprises:

[0149] In a further aspect, provided herein is an antibody that immunospecifically binds to human KIT, (i) Amino acid sequence: [ka] (where X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K3 is an amino acid having an aliphatic hydroxyl side chain, and X K4 is an amino acid having an aliphatic hydroxyl side chain or is P; X K5is an amino acid having a charged or acidic side chain, and X K6 is an amino acid having an aromatic side chain; [ka] where X H1 is an amino acid having an aliphatic side chain, and X H2 is an amino acid having an aliphatic side chain, and X H3 is an amino acid with a polar or basic side chain, and X H4 is an amino acid having an aliphatic side chain, and X H5 is an amino acid having an aliphatic side chain, and X H6 is an amino acid with an acidic side chain, and X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain) and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain that also contains the unnatural amino acids 234A, 235Q, 322Q, and preferably 252Y, 254T, and 256E, as numbered by the EU index as set forth in Kabat.

[0150] In a further aspect, provided herein is an antibody that immunospecifically binds to human KIT, comprising: i) a VL comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16, and ii) a VH comprising the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain that also contains unnatural amino acids 234A, 235Q, 322Q, and preferably 252Y, 254T, and 256E, as numbered by the EU index as set forth in Kabat.

[0151] In a further aspect, provided herein is an antibody that immunospecifically binds to human KIT, comprising: i) a VL comprising the amino acid sequence of SEQ ID NO: 14; and ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain that also contains unnatural amino acids 234A, 235Q, 322Q, and preferably 252Y, 254T, and 256E, as numbered by the EU index as set forth in Kabat.

[0152] In a specific embodiment, the antibody provided herein has the following amino acid sequence: [ka] The heavy chain comprises:

[0153] In a specific embodiment, the antibody provided herein has the following amino acid sequence: [ka] The light chain comprises

[0154] In a specific embodiment, the antibody provided herein has the following amino acid sequence: [ka] and a heavy chain comprising the amino acid sequence: [ka] The light chain comprises

[0155] In specific embodiments, provided herein is (i) Amino acid sequence: [ka] Here, the leader sequence is shown in bold italics, the variable region (VH) is shown in italics, and the constant region is underlined. Additionally, mutations within the constant region (relative to wild-type human IgG1) are double underlined. a heavy chain comprising (ii) Amino acid sequence: [ka] (wherein the leader sequence is shown in bold italics, the variable region (VL) is shown in italics, and the constant region is underlined) and a light chain comprising:

[0156] In a specific embodiment, the anti-KIT antibody described herein does not bind to any human Fc gamma receptor (FcγR receptor) (e.g., has no detectable binding). In a specific embodiment, the anti-KIT antibody described herein does not bind to human FcγRI (e.g., has no detectable binding). In a specific embodiment, the anti-KIT antibody described herein does not bind to human FcγRIIa (e.g., has no detectable binding). In a specific embodiment, the anti-KIT antibody described herein does not bind to human FcγRIIb (e.g., has no detectable binding). In a specific embodiment, the anti-KIT antibody described herein does not bind to human FcγRIIIa (e.g., has no detectable binding). In a specific embodiment, the anti-KIT antibody described herein does not bind to human FcγRIIIb (e.g., has no detectable binding).

[0157] In a specific embodiment, the anti-KIT antibody described herein comprises a modified (e.g., mutated) human IgG constant region (e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 constant region) and has enhanced binding (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, or 10000-fold higher binding affinity) to human fetal Fc receptor (FcRn) compared to a corresponding antibody having the same variable region sequence but an unmodified (wild-type) human IgG constant region. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 20 nM at pH 6.0. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 2 nM at pH 6.0. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 1 nM at pH 6.0. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 500 nM at pH 6.0. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 400 pM at pH 6.0. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 200 nM at pH 7.2. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 150 nM at pH 7.2. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 100 nM at pH 7.2. In a specific embodiment, the anti-KIT antibody described herein binds to FcRn with a KD of less than 80 nM at pH 7.2.

[0158] In a specific embodiment, the anti-KIT antibodies described herein comprise a modified (e.g., mutated) human IgG constant region (e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 constant region) and do not exhibit antibody-dependent cellular cytotoxicity (ADCC). In a specific embodiment, the anti-KIT antibodies described herein comprise a modified (e.g., mutated) human IgG constant region (e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 constant region) and exhibit reduced ADCC (e.g., at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less) compared to a corresponding antibody having the same variable region sequence but an unmodified (wild-type) human IgG constant region.

[0159] In a specific embodiment, the anti-KIT antibodies described herein comprise a modified (e.g., mutated) human IgG constant region (e.g., a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 constant region) and exhibit reduced cytokine (e.g., IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-10, and / or TNF-α) production compared to a corresponding antibody having the same variable region sequence but an unmodified (wild-type) human IgG constant region (e.g., at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less).

[0160] In specific embodiments, also provided are antigen-binding fragments of the antibodies described herein, preferably antigen-binding fragments that comprise a full-length heavy chain Fc region or domain (e.g., full-length human IgG1, full-length human IgG2, full-length human IgG3, or full-length human IgG4 Fc region or domain). In specific embodiments, also provided are antigen-binding fragments of the antibodies described herein that comprise a partial heavy chain Fc region or domain (e.g., partial human IgG1, partial human IgG2, partial human IgG3, or partial human IgG4 Fc region or domain).

[0161] In certain embodiments, anti-KIT antibodies or antigen-binding fragments thereof can be obtained using methods known in the art, see, e.g., Section 5.4 below.

[0162] In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to the D4 domain of human KIT and to KIT, e.g., the D5 region of human KIT. In another specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to KIT, e.g., the D5 domain of human KIT, with a lower affinity than to the D4 domain of KIT, e.g., human KIT. In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to KIT, e.g., the D4 domain of human KIT, with a higher affinity than to the D5 domain of KIT, e.g., human KIT; for example, the higher affinity is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold, as determined by methods known in the art, e.g., ELISA or Biacore assay.

[0163] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the D4 or D4 / D5 region of KIT, e.g., human KIT, and have an affinity for a KIT antigen consisting essentially of only the D4 domain that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher than a KIT antigen consisting essentially of only the D5 domain.

[0164] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein have an EC of about 50 nM, 10 nM, 500 pM, 300 pM, 200 pM, 100 pM, or 50 pM or less as determined by assays described in the art, such as ELISA. 50 (half maximal effective concentration) value to specifically bind to a KIT polypeptide (eg, the D4 domain of human KIT).

[0165] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein have an EC of about 200 pM or 150 pM or less as determined by assays described in the art, such as ELISA or FACs using CHO-WT-KIT cells (CHO cells recombinantly modified to express wild-type human KIT). 50 The antibody specifically binds to a KIT polypeptide (eg, the D4 region of human KIT) at a value

[0166] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein have an IC of about 600 pM or less. 50 (50% inhibitory concentration) value capable of blocking KIT phosphorylation.

[0167] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor internalization, e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as compared to internalization in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to one of skill in the art. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor internalization, e.g., by at least about 25% or 35%, optionally up to about 75%, as compared to internalization in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to one of skill in the art. In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein can induce or enhance KIT receptor internalization, for example, at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as compared to internalization in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those skilled in the art. Techniques for quantifying or visualizing cell surface receptors are well known in the art and include various fluorescent and radioactive techniques. For example, one method involves incubating cells with a radiolabeled anti-receptor antibody. Alternatively, the natural ligand of the receptor can be conjugated to a fluorescent molecule or a radioactive label and incubated with the cells.Additional receptor internalization assays are known in the art and are described, for example, in Jimenez et al., Biochemical Pharmacology, 1999, 57:1125-1131; Bernhagen et al., Nature Medicine, 2007, 13:587-596; and Conway et al., J. Cell Physiol., 2001, 189:341-55.

[0168] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor turnover, e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as compared to turnover in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT) when assessed by methods described herein or known to one of skill in the art. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor turnover by at least about 25% or 35%, optionally up to about 75%, as compared to turnover in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT) when assessed by methods described herein or known to one of skill in the art. In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein can induce or enhance KIT receptor turnover by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as compared to turnover in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art. Methods for determining receptor turnover are well known in the art. For example, cells expressing KIT can be cultured in a 50-well plate, ... 35Cells can be pulse-labeled with S-EXPRESS Protein Labeling Mix (NEG772, NEN Life Science Products), washed, and chased with non-labeling medium for a period of time, after which protein lysates from the labeled cells are immunoprecipitated with anti-KIT antibodies, separated by SDS-PAGE, and visualized (e.g., exposed to a PhosphoImager screen (Molecular Dynamics), scanned with a Typhoon 8600 scanner (Amersham), and analyzed with ImageQuant software (Molecular Dynamics)) (see, e.g., Chan et al., Development, 2004, 131:5551-5560).

[0169] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor degradation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to degradation in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT) when assessed by methods described herein or known to one of skill in the art. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor degradation by at least about 25% or 35%, optionally up to about 75%, compared to degradation in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT) when assessed by methods described herein or known to one of skill in the art. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can induce or enhance KIT receptor degradation by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as compared to degradation in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to one of skill in the art. Techniques for quantifying or monitoring the ubiquitination and / or degradation (e.g., kinetics or rate of degradation) of cell surface receptors are well known in the art and include a variety of fluorescent and radioactive techniques (see, e.g., International Patent Application Publication No. WO 2008 / 153926 A2). For example, pulse-chase experiments or 125 Experiments using radiolabeled ligands such as I-SCF can be performed to quantitatively measure the degradation of KIT.

[0170] In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein does not bind to the extracellular ligand binding site of KIT, for example, the SCF binding site of KIT. In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein does not inhibit ligand binding to KIT, for example, does not inhibit KIT ligand (e.g., SCF) binding to KIT, as determined by methods described in the art, for example, ELISA. In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein does not completely or partially inhibit ligand binding to KIT, for example, does not completely or partially inhibit KIT ligand (e.g., SCF) binding to KIT, as determined by methods described in the art, for example, ELISA or FACS (fluorescence-activated cell sorting).

[0171] In specific aspects, the anti-KIT antibodies (e.g., human or humanized antibodies) provided herein are inhibitory antibodies, i.e., antibodies that inhibit (e.g., partially inhibit) KIT activity, i.e., one or more KIT activities. In specific embodiments, partial inhibition of KIT activity results in, for example, about 25% to about 65% or 75% inhibition. In specific embodiments, partial inhibition of KIT activity results in, for example, about 35% to about 85% or 95% inhibition. Non-limiting examples of KIT activities include KIT dimerization, KIT phosphorylation (e.g., tyrosine phosphorylation), signaling downstream of KIT (e.g., Stat, AKT, MAPK, or Ras signaling), induction or enhancement of gene transcription (e.g., c-Myc), induction or enhancement of cell proliferation, or cell survival. In certain embodiments, the antibodies described herein inhibit KIT phosphorylation (e.g., ligand-induced phosphorylation).

[0172] In a specific embodiment, the anti-KIT antibodies, or antigen-binding fragments thereof, provided herein inhibit KIT tyrosine phosphorylation in the KIT cytoplasmic domain.

[0173] In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit cell proliferation, e.g., mast cell proliferation or eosinophil proliferation. In yet another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit cell survival, e.g., mast cell survival or eosinophil cell survival. In some embodiments, the inhibition of cell proliferation, e.g., mast cell proliferation or eosinophil proliferation, is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0174] In another specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits mast cell activation or eosinophil activation. In certain embodiments, inhibition of mast cell activation or activity or eosinophil activation or activity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0175] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit eosinophil or mast cell degranulation (see, e.g., Staats et al., 2012, Med. Chem. Commun., 2013, 4:88-94; and Ochkur et al., 2012, J. Immunol. Methods, 384: 10-20). In some embodiments, inhibition of eosinophil or mast cell degranulation is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0176] In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit the release of mast cell mediators. In certain embodiments, the release of mast cell mediators is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Assays for measuring mast cell activity, such as mediator release, from mast cell cultures, such as rodent and human mast cell cultures, have been described (see, e.g., Kuehn et al., "Measuring Mast Cell Mediator Release," Current Protocols in Immunology, Unite 7.38.1- 7.38.9, November 2010 (John Wiley & Sons, Inc.)). In certain embodiments, CD34 peripheral blood progenitor cells or mast cell lines, such as HMC-1 or human LAD2 mast cell lines, can be used in these assays to confirm the effect of anti-KIT antibodies on mast cells.

[0177] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein induce apoptosis, e.g., mast cell apoptosis or eosinophil apoptosis. In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein induce cell differentiation, e.g., mast cell differentiation.

[0178] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of achieving any one of the following: reducing eosinophil numbers and / or activity, reducing mast cell proliferation, reducing plasma tryptase levels, reducing plasma SCF levels, reducing mast cell numbers or amounts, inhibiting or reducing mast cell activity, reducing mast cell-induced production or release of inflammatory factors, reducing release of inflammatory factors, restoring mast cell homeostasis, reducing mast cell migration, reducing mast cell adhesion, inhibiting or reducing mast cell recruitment of eosinophils, and inhibiting or reducing antigen-mediated degranulation of mast cells.

[0179] In certain embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT activity but does not inhibit KIT dimerization. In another specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT activity but does not inhibit ligand binding to KIT, for example, does not inhibit KIT ligand (e.g., SCF) binding to KIT but inhibits KIT dimerization.

[0180] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity, e.g., ligand-induced tyrosine phosphorylation of the KIT cytoplasmic domain, by about 25% to about 65% or 75%, as determined by a cell-based phosphorylation assay well known in the art, e.g., a cell-based phosphorylation assay described herein. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity, e.g., ligand-induced tyrosine phosphorylation of the KIT cytoplasmic domain, by about 35% to about 85% or 95%, as determined by a cell-based phosphorylation assay well known in the art, e.g., a cell-based phosphorylation assay described herein.

[0181] In certain embodiments, the anti-KIT antibodies, or antigen-binding fragments thereof, provided herein have a 50% inhibitory concentration (IC) of less than about 600 pM, or less than about 500 pM, or less than about 250 pM, as determined by a cell-based phosphorylation assay well known in the art, such as a cell-based phosphorylation assay described herein. 50 ) inhibits KIT activity, e.g., ligand-induced tyrosine phosphorylation of the KIT cytoplasmic domain. In a specific embodiment, the IC 50 In certain embodiments, the IC 50 In a specific embodiment, the IC is in the range of about 50 pM to about 225 pM, or in the range of 100 pM to about 600 pM. 50 is in the range of about 50 pM to about 550 pM, or about 50 pM to about 600 pM, or about 150 pM to about 550 pM.

[0182] In a specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein (i) immunospecifically binds to a KIT polypeptide comprising the D4 and / or D5 region of human KIT, (ii) inhibits KIT phosphorylation (e.g., tyrosine phosphorylation), and (iii) does not completely or partially inhibit KIT ligand (e.g., SCF) binding to KIT. In yet another specific embodiment, such an antibody does not inhibit KIT dimerization. In yet another specific embodiment, such an antibody can be recombinantly expressed by CHO cells with an average titer of at least 0.5 μg / mL, e.g., at least 1.0 μg / mL. In a further specific embodiment, such an antibody comprises a VH domain and a VL domain that are non-immunogenic, e.g., the VH domain and the VL domain do not contain a T cell epitope.

[0183] In other specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein immunospecifically bind to the monomeric form of KIT (e.g., human KIT). In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the monomeric form of KIT (e.g., human KIT). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the dimeric form of KIT (e.g., human KIT).

[0184] In a specific embodiment, the anti-KIT antibody or its antigen-binding fragment provided herein does not bind to KIT in monomeric form, but specifically binds to KIT in dimeric form or KIT in multimeric form.In some embodiments, the antibody has a higher affinity for KIT monomer than KIT dimer.In some embodiments, the antibody has a higher affinity for KIT monomer than KIT multimer.

[0185] In a specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to a native isoform or native variant of KIT (i.e., a naturally occurring isoform or variant of KIT in an animal (e.g., monkey, mouse, goat, donkey, dog, cat, rabbit, pig, rat, human, frog, or bird) that can be isolated from an animal, preferably a human). In a specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to human KIT or a fragment thereof. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to human KIT or a fragment thereof and does not specifically bind to non-human KIT (e.g., monkey, mouse, goat, donkey, dog, cat, rabbit, pig, rat, or bird) or a fragment thereof. In a specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to human KIT or a fragment thereof and does not specifically bind to mouse KIT. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof (e.g., the D4 region of human KIT) and canine (dog) and non-human primate (e.g., monkey) KIT. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof (e.g., the D4 region of human KIT) and canine (dog) and non-human primate (e.g., monkey) KIT, but do not specifically bind to mouse or rat KIT or a fragment thereof (e.g., the D4 region of mouse KIT).

[0186] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof (e.g., the D4 region of human KIT) and canine (dog), feline (cat), and cynomolgus KIT, but do not specifically bind to mouse or rat KIT or a fragment thereof (e.g., the D4 region of mouse KIT).

[0187] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof (e.g., the D4 region of human KIT) and canine (dog), feline (cat), and cynomolgus KIT with greater affinity (e.g., at least 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold) than to mouse or rat KIT or a fragment thereof (e.g., the D4 region of mouse KIT).

[0188] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the extracellular domain of human KIT that contains a mutation, e.g., a somatic mutation, e.g., a mutation in exon 9 of human KIT that duplicates Ala and Tyr residues at positions 502 and 503 (see, e.g., Marcia et al. (2000) Am. J. Pathol. 156(3):791-795; and Debiec-Rychter et al. (2004) European Journal of Cancer. 40:689-695, which describe KIT mutations, both of which are incorporated herein by reference in their entireties).

[0189] In some embodiments, the anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to the extracellular domain of human KIT that is glycosylated. In some embodiments, the antibody or antigen-binding fragment thereof described herein binds to two different glycosylated forms of the extracellular domain of human KIT. For example, two forms of human KIT with different molecular weights that show different glycosylation patterns have been observed by immunoblotting.

[0190] In certain embodiments, the antibodies described herein can specifically bind to both of these forms of human KIT, which have different glycosylation patterns, e.g., one form is more glycosylated than the other. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the extracellular domain of unglycosylated human KIT.

[0191] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are bivalent monospecific antibodies in that they have two antigen-binding regions (e.g., two identical antigen-binding regions) and both antigen-binding regions specifically bind to the same antigen, KIT (e.g., human KIT). In certain embodiments, the antigen-binding regions comprise the VH and VL CDRs set forth in Table 1. In certain embodiments, the antigen-binding regions comprise a VH comprising the amino acid sequence of any one of SEQ ID NOs: 8-12, and / or a VL comprising the amino acid sequence of any one of SEQ ID NOs: 13-16. In certain aspects, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are not bispecific antibodies.

[0192] In a specific embodiment, the antibody described herein is a monoclonal antibody or an isolated monoclonal antibody. In another specific embodiment, the antibody described herein is a humanized monoclonal antibody. In a particular embodiment, the antibody described herein is a recombinant antibody, such as a recombinant human antibody, a recombinant humanized antibody, or a recombinant monoclonal antibody. In some embodiments, the antibody described herein contains non-human amino acid sequences, such as non-human CDRs or non-human (e.g., non-human primate) framework residues.

[0193] In certain embodiments provided herein, the recombinant antibody can be isolated, prepared, expressed, or produced by recombinant means, including, for example, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial antibody library, or antibodies prepared, expressed, produced, or isolated by any other means, including, for example, synthesis, genetic modification of DNA sequences encoding human immunoglobulin sequences, or production by splicing of human immunoglobulin-encoding sequences, e.g., human immunoglobulin gene sequences, to other such sequences. In certain embodiments, the amino acid sequences of such recombinant antibodies are so modified that the amino acid sequences of such antibodies, e.g., the VH and / or VL regions, are sequences that do not naturally occur in the in vivo antibody germline repertoire of an organism, e.g., the mouse or human germline repertoire. In certain embodiments, a recombinant antibody can be obtained by assembling several sequence fragments that are naturally occurring in an organism (e.g., a primate, e.g., a human) into a composite sequence of a recombinant antibody, where the composite sequence does not naturally occur in the organism (e.g., a primate, e.g., a human).

[0194] The antibodies provided herein include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. In a specific embodiment, the antibodies provided herein are IgG antibodies (e.g., human IgG antibodies), or a class (e.g., human IgG1 or IgG4) or subclass thereof. In another specific embodiment, the antibodies described herein are IgG1 (e.g., human IgG1 (isotype a, z, or f)) or IgG4 antibodies. In certain embodiments, the antibodies described herein are whole or entire antibodies, e.g., whole or entire humanized, human, or composite human antibodies.

[0195] In a specific embodiment, the antibody provided herein comprises an antibody light chain and a heavy chain, e.g., separate light and heavy chains. With respect to the light chain, in a specific embodiment, the light chain of the antibody described herein is a kappa light chain. In another specific embodiment, the light chain of the antibody described herein is a lambda light chain. In yet another specific embodiment, the light chain of the antibody described herein is a human kappa light chain or a human lambda light chain. In a particular embodiment, the antibody described herein comprises a human light chain constant region. Non-limiting examples of human light chain constant region sequences are described in the art, see, for example, U.S. Patent No. 5,693,780 and Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242.

[0196] With respect to the heavy chain, in specific embodiments, the heavy chain of the antibodies described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In another specific embodiment, the heavy chain of the antibodies described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In certain embodiments, the antibodies described herein comprise a human heavy chain constant region (e.g., a human IgG constant region, e.g., a human IgG1, IgG2, IgG3, or IgG4 constant region). Non-limiting examples of human heavy chain constant region sequences are described in the art, see, for example, U.S. Patent No. 5,693,780 and Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. In specific embodiments, the antibodies described herein comprise a modified (e.g., mutated) human Fc region or domain (e.g., a modified (e.g., mutated) human IgG1 Fc region or domain, a modified (e.g., mutated) human IgG2 Fc region or domain, a modified (e.g., mutated) human IgG3 Fc region or domain, or a modified (e.g., mutated) human IgG4 Fc region or domain).

[0197] In certain embodiments, the anti-KIT antibodies described herein are human, bihuman, or humanized monoclonal antibodies. In certain embodiments, the antibodies described herein are engineered antibodies, e.g., recombinantly produced antibodies. In specific embodiments, the antibodies described herein are humanized antibodies comprising one or more non-human (e.g., rodent or mouse) CDRs and one or more human framework regions (FRs), and optionally, human heavy chain constant regions and / or light chain constant regions. In specific embodiments, the antibodies described herein comprise one or more primate (or non-human primate) framework regions. In specific embodiments, the antibodies described herein do not comprise non-human primate framework regions.

[0198] The antibodies provided herein may include antibodies that include chemical modifications, e.g., antibodies that are chemically modified, e.g., by the covalent attachment of any type of molecule to the antibody. For example, but not limited to, the anti-KIT antibodies may be glycosylated, acetylated, pegylated, phosphorylated, or amidated, may be derivatized with a protecting / blocking group, or may further include a cellular ligand and / or other protein or peptide (e.g., a heterologous protein or peptide), and the like. For example, the antibodies provided herein may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, and the like. Additionally, the anti-KIT antibodies described herein may contain one or more non-classical amino acids.

[0199] In one embodiment, the anti-KIT antibody provided herein is a naked antibody that is not linked, fused or conjugated (e.g., artificially linked, fused or conjugated) to another molecule, peptide or polypeptide (e.g., a heterologous polypeptide). In certain embodiments, the anti-KIT antibody provided herein is not an antibody-drug conjugate. In certain embodiments, the anti-KIT antibody provided herein is not a fusion protein. In certain embodiments, the anti-KIT antibody described herein does not comprise a non-classical amino acid.

[0200] 5.1.1. Conjugates In some embodiments, provided herein are antibodies (e.g., human or humanized antibodies) conjugated or recombinantly fused to diagnostic, detection or therapeutic agents or any other molecules, or antigen-binding fragments thereof. Conjugated or recombinantly fused antibodies may be useful, for example, for monitoring or prognosing the occurrence, development, progression, and / or severity of KIT-related disorders or diseases, for example, as part of a clinical trial procedure, such as determining the efficacy of a particular therapy. Conjugated or recombinantly fused antibodies may be useful, for example, for treating or managing KIT-related disorders, or for preventing, treating or managing the effects of KIT-related disorders. The antibodies described herein may also be conjugated to a molecule (e.g., polyethylene glycol) that may affect one or more biological and / or molecular properties of the antibody, such as stability (e.g., in serum), half-life, solubility, and antigenicity.

[0201] In certain aspects, provided herein is a conjugate comprising an agent (e.g., a therapeutic agent) linked to an antibody (or antigen-binding fragment thereof) described herein. In specific embodiments, the conjugate comprises an antibody described herein and a molecule (e.g., a therapeutic or drug moiety), the antibody being linked to the molecule directly or via one or more linkers. In certain embodiments, the antibody is covalently conjugated to the molecule. In certain embodiments, the antibody is non-covalently conjugated to the molecule. In specific embodiments, the antibody described herein, e.g., the antibody conjugated to the agent, binds to wild-type human KIT. In certain embodiments, the antibody described herein, e.g., the antibody conjugated to the agent, binds to the extracellular domain of human KIT that contains a mutation, e.g., a somatic mutation associated with cancer (e.g., GIST), e.g., a mutation in exon 9 of human KIT that duplicates Ala and Tyr residues at positions 502 and 503.

[0202] Such diagnosis and detection can be accomplished, for example, by coupling the antibody to a detectable molecule or substance, including various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as, but not limited to, iodine ( 131 I, 125 I, 123 I, and 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In, and 111 In), Technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), Molybdenum ( 99 Mo), Xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117 Sn; as well as positron emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions.

[0203] Provided are antibodies or antigen-binding fragments thereof described herein conjugated or recombinantly fused to a therapeutic moiety (or one or more therapeutic moieties), and uses of such antibodies. The antibodies can be conjugated or recombinantly fused to a therapeutic moiety, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, e.g., an α-emitter. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Therapeutic moieties include auristatins or derivatives thereof, such as monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin PYE, and auristatin E (AE) (see, e.g., U.S. Pat. No. 7,662,387 and U.S. Patent Application Publication Nos. 2008 / 0300192 and 2008 / 0025989, each of which is incorporated herein by reference); microtubule disrupting agents, such as maytansine or derivatives thereof, such as the maytansinoid DM1 (see, e.g., U.S. Pat. Nos. 7,851,432, 7,575,748, and 5,416,064, each of which is incorporated herein by reference); prodrugs, such as prodrugs of CC-1065 (rachelmycin) analogs; metabolic antagonists (e.g., methotrexate, 6-mer, captopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP), and cisplatin); minor groove-binding alkylating agents; anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)); auristatin molecules (e.g., auristatin PHE, bryostatin 1, and solastatin 10;Woyke et al., Antimicrob. Agents Chemother. 46:3802-8 (2002); Woyke et al., Antimicrob. Agents Chemother. 45:3580-4 (2001); Mohammad et al., Anticancer Drugs 12:735-40 (2001); Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999); Mohammad et al., Int. J. Oncol. 15:367-72 (1999), all of which are incorporated herein by reference; hormonal agents (e.g., glucocorticoids, progestins, androgens, and estrogens), DNA repair enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8(7):2167-76 (2002));Cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof, and the compounds described in U.S. Pat. Nos. 6,245,759, 6,399,633, 6,421,522, and 6,521,522) may be used. No. 6,383,790, No. 6,335,156, No. 6,271,242, No. 6,242,196, No. 6,218,410, No. 6,218,3 No. 72, No. 6,057,300, No. 6,034,053, No. 5,985,877, No. 5,958,769, No. 5,925,376, No. 5,922, 844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, and 5,587,459, each of which is incorporated herein by reference for its disclosure of such compounds;Farnesyltransferase inhibitors (e.g., R115777, BMS-214662, as well as those described in, for example, U.S. Patents: 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,39 No. 9,615, No. 6,387,905, No. 6,372,747, No. 6,369,034, No. 6,362,188, No. 6,342,765, No. 6,342,487, No. 6,300,501 , No. 6,268,363, No. 6,265,422, No. 6,248,756, No. 6,239,140, ​​No. 6,232,338, No. 6,228,865, No. 6,228,856, No. 6,22 No. 5,322, No. 6,218,406, No. 6,211,193, No. 6,187,786, No. 6,169,096, No. 6,159,984, No. 6,143,766, No. 6,133,303 No. 6,127,366, No. 6,124,465, No. 6,124,295, No. 6,103,723, No. 6,093,737, No. 6,090,948, No. 6,080,870, No. 6,0 77,853, 6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305, each of which is incorporated herein by reference for its disclosure of such inhibitors); topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000; sientopine; UCE6; UCE1022; TAN-1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and rebeccamycin); bulgarein; DNA minor groove binders, such as Hoescht dye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine; coraline;β-lapachone; BC-4-1; bisphosphonates (e.g., alendronate, cimadronte, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zoledronate), HMG-CoA reductase inhibitors (e.g., lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statins, cerivastatin, lescol, lupitor, rosuvastatin, and atorvastatin); antisense oligonucleotides (e.g., U.S. Pat. No. 6,233,633; Nos. 77,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709, each of which is incorporated herein by reference with respect to such oligonucleotides; adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) and pharma- ceutically acceptable salts, solvates, clathrates, and prodrugs thereof;

[0204] In certain embodiments, the therapeutic or drug moiety is an antitubulin drug, such as auristatin or its derivative.Non-limiting examples of auristatins include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PYE, and auristatin E (AE) (see, for example, U.S. Patent No. 7,662,387 and U.S. Patent Application Publication Nos. 2008 / 0300192 and 2008 / 0025989, each of which is incorporated herein by reference).In some embodiments, the therapeutic or drug moiety is a microtubule disrupting agent, such as maytansine or its derivative, such as maytansinoid DM1 or DM4 (see, for example, U.S. Patent Nos. 7,851,432, 7,575,748, and 5,416,064, each of which is incorporated herein by reference). In certain embodiments, the therapeutic moiety or drug moiety is a prodrug, e.g., a prodrug of a CC-1065 (rachelmycin) analog (see, e.g., U.S. Patent Application Publication No. 2008 / 0279868, and PCT International Patent Application Publication Nos. WO 2009 / 017394, WO 2010 / 062171, and WO 2007 / 089149, each of which is incorporated herein by reference).

[0205] In a specific embodiment, the antibody and therapeutic agent / drug substance are conjugated by one or more linkers. In another specific embodiment, the antibody and therapeutic agent / drug substance are directly conjugated.

[0206] In specific embodiments, non-limiting examples of therapeutic or drug moieties for conjugation to the antibodies described herein include calicheamicin (e.g., LL-E33288 conjugates, e.g., gamma-calicheamicin, see, e.g., U.S. Pat. No. 4,970,198) and derivatives thereof (e.g., gamma calicheamicin hydrazide derivatives), ozogamicin, duocarmycins and their derivatives (e.g., CC-1065 (NSC 298223), or achiral analogs of duocarmycins (e.g., AS-1-145 or centanamycin)), taxanes and their derivatives, and enediynes and their derivatives (see, e.g., PCT International Patent Publication Nos. WO 2009 / 017394, WO 2010 / 062171, WO 2007 / 089149, WO 2011 / 021146, WO See WO 2008 / 150261, WO 2006 / 031653, WO 2005 / 089809, WO 2005 / 089807, and WO 2005 / 089808, each of which is incorporated by reference in its entirety.

[0207] Non-limiting examples of calicheamicins suitable for conjugation to the antibodies described herein are disclosed in, e.g., U.S. Pat. Nos. 4,671,958; 5,053,394; 5,037,651; 5,079,233; and 5,108,912; and PCT International Patent Application Publication Nos. WO 2011 / 021146, WO 2008 / 150261, WO 2006 / 031653, WO 2005 / 089809, WO 2005 / 089807, and WO 2005 / 089808; each of which is incorporated by reference herein for its disclosure of such calicheamicins. In certain embodiments, these compounds may contain methyl trisulfides that react with appropriate thiols to form disulfides while simultaneously introducing functional groups such as hydrazides or other functional groups that may be useful for conjugating calicheamicin to the antibodies described herein. In certain embodiments, stabilizing the disulfide bonds present in the calicheamicin conjugates by adding dimethyl substituents may result in improved antibody / drug conjugates. In a specific embodiment, the calicheamicin derivative is N-acetylgamma calicheamicin dimethylhydrazide, or NAc-gamma DMH (CL-184,538), as one of the optimized derivatives for conjugation. Disulfide analogs of calicheamicin that can be conjugated to the antibodies described herein are described, for example, in U.S. Patent Nos. 5,606,040 and 5,770,710, each of which is incorporated herein by reference for its disclosure of such compounds. In certain embodiments, a moiety (e.g., calicheamicin or a derivative thereof) is conjugated to an antibody by a linker. In certain embodiments, the moiety (e.g., calicheamicin or a derivative thereof) is hydrolyzed from the antibody-drug conjugate at the linker. In one embodiment, the moiety (e.g., calicheamicin or a derivative thereof) is hydrolyzed from the antibody conjugate at the linker at about pH 3.0 to pH 4.0, for 1 to 24 hours, at a temperature of 20 to 50° C., preferably 37° C.

[0208] In specific embodiments, non-limiting examples of therapeutic or drug moieties for conjugation to the antibodies described herein include pyrrolobenzodiazepines (PBDs) and derivatives thereof, such as PBD dimers (e.g., SJG-136 or SG2000), C2-unsaturated PBD dimers, pyrrolobenzodiazepine dimers with C2 aryl substitutions (e.g., SG2285), hydrolytically activated PBD dimer prodrugs (e.g., SG2285), and polypyrrole-PBDs (e.g., SG2274) (see, e.g., PCT International Patent Application Publication Nos. WO 2000 / 012507, WO 2007 / 039752, WO 2005 / 110423, WO 2005 / 085251, and WO No. 2005 / 040170, as well as U.S. Pat. No. 7,612,062, each of which is incorporated herein by reference for its disclosure of such compounds.

[0209] Additionally, the antibodies described herein can be coupled to a therapeutic moiety, e.g., a radioactive metal ion, e.g., an α-emitter, e.g., 213 Bi, or, but not limited to, 131 In, 131 LU, 131 Y, 131 Ho, 131 The macrocyclic chelator can be conjugated to a macrocyclic chelator useful for coupling radiometal ions, including Sm, to polypeptides. In one embodiment, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each of which is fully incorporated by reference.

[0210] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are conjugated to one or more molecules (e.g., therapeutic or drug moieties) directly or indirectly via one or more linker molecules. In certain embodiments, the linker is an enzyme-cleavable linker or a disulfide linker. In specific embodiments, the cleavable linker is cleavable by an enzyme, such as an aminopeptidase, an aminoesterase, a dipeptidyl carboxypeptidase, or a protease of the blood coagulation cascade. In certain embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acid residues. In certain embodiments, the linker consists of 1 to 10 amino acid residues, 1 to 15 amino acid residues, 5 to 20 amino acid residues, 10 to 25 amino acid residues, 10 to 30 amino acid residues, or 10 to 50 amino acid residues.

[0211] In certain embodiments, the moiety is conjugated to the antibody by one or more linkers. In certain embodiments, the moiety is hydrolyzed from the antibody-drug conjugate at the linker. In one embodiment, the moiety is hydrolyzed from the antibody conjugate at the linker at about pH 3.0 to pH 4.0, for 1 to 24 hours, and at a temperature of 20 to 50°C, preferably 37°C. In a specific embodiment, the linker is stable in the bloodstream, but releases the conjugated moiety once it enters the interior of a target cell. In certain embodiments, the moiety is conjugated to the antibody described herein via one or more triazole-containing linkers (see, for example, International Patent Application Publication No. WO 2007 / 018431, which is incorporated herein by reference). Non-limiting examples of linkers and spacers for incorporation into the antibody-drug conjugates described herein are disclosed in PCT International Patent Application Publication Nos. WO 2007 / 018431, WO 2004 / 043493, and WO 2002 / 083180.

[0212] Additionally, the antibodies described herein can be fused to a marker sequence, e.g., a peptide to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, many of which are commercially available, such as the tag provided in the pQE vector (QIAGEN, Inc.), among others. For example, hexa-histidine provides for convenient purification of the fusion protein, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "FLAG" tag.

[0213] Methods of fusing or conjugating therapeutic moieties (including polypeptides) to antibodies are well known and can be found, for example, in Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., Antibodies For Drug Delivery, Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, 1997. Review," Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); Thorpe et al., 1982, Immunol. Rev.62:119-58; U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT Publication Nos. WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; See Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol., 154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA, 89:11337-11341, 1992, which are incorporated by reference in their entireties.

[0214] The antibodies described herein can also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0215] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are extracellular drug conjugates (ECDs) that include an antibody linked to a drug, optionally by a linker (see, e.g., PCT International Patent Application Publication No. WO 2011 / 031870). The drug can act extracellularly, and therefore internalization of the conjugate is not required. After the ECD binds to the target cell, the drug sends a signal into the cell.

[0216] In one embodiment, the linker of ECD is a non-cleavable linker.Examples of non-cleavable linkers include linkers containing polyethylene glycol or polyethylene chains that are not acid or base sensitive (e.g., hydrazone-containing linkers), are not sensitive to reducing or oxidizing agents (e.g., those containing disulfide bonds), and are not sensitive to enzymes that can be found in cells or the circulatory system.Specific examples of non-cleavable linkers include SMCC linkers (US Patent Application No. 20090202536). To illustrate, examples of cleavable linkers include linkers containing unhindered glutathione-sensitive disulfides, esters, peptide sequences sensitive to peptidases such as cathepsin or plasmin, pH-sensitive hydrazones (see Bioconjugate Chem., 2010, 21(1), pp 5-13) and the unhindered disulfide linker SPP (see U.S. Patent Application No. 20090202536).

[0217] In some embodiments, the ECD comprises a drug or agent that is a cardiac glycoside, e.g., proscillaridin or sugar-enhanced proscillaridin. In one embodiment, the agent is comprised of a cardiac glycoside that lacks sugar. In various embodiments, the cardiac glycoside is a compound identified in PCT Publication WO 2010 / 017480 (PCT / US2009 / 053159).

[0218] 5.2 Polynucleotides In certain aspects, provided herein are polynucleotides and combinations of polynucleotides that comprise a nucleotide sequence(s) encoding an antibody (e.g., a human or humanized antibody) or a fragment thereof (e.g., a variable light chain region and / or a variable heavy chain region) described herein that immunospecifically binds to a KIT antigen. Also provided herein are polynucleotides encoding the KIT antigen for making the anti-KIT antibodies described herein.

[0219] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., human). Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, can be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In a specific embodiment, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0220] In a particular aspect, provided herein is a polynucleotide or a combination of polynucleotides comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described herein, or a VH and VL of said antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein is a polynucleotide comprising a nucleotide sequence encoding the VH of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein is a polynucleotide comprising a nucleotide sequence encoding the VL of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein is a polynucleotide comprising a first nucleotide sequence encoding the VH of an antibody or antigen-binding fragment thereof described herein, and a second nucleotide sequence encoding the VL of said antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein is a combination of two polynucleotides, the first polynucleotide of the combination comprising a first nucleotide sequence encoding the VH of an antibody or antigen-binding fragment thereof described herein, and the second polynucleotide of the combination comprising a second nucleotide sequence encoding the VL of said antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein is a polynucleotide comprising a nucleotide sequence encoding the heavy chain of an antibody described herein. In a specific embodiment, provided herein is a polynucleotide comprising a nucleotide sequence encoding the light chain of an antibody described herein. In a specific embodiment, provided herein is a polynucleotide comprising a first nucleotide sequence encoding the heavy chain of an antibody described herein and a second nucleotide sequence encoding the light chain of said antibody. In a specific embodiment, provided herein is a combination of two polynucleotides, wherein a first polynucleotide of the combination comprises a first nucleotide sequence encoding the heavy chain of an antibody described herein and a second polynucleotide of the combination comprises a second nucleotide sequence encoding the light chain of said antibody.

[0221] In a specific embodiment, the polynucleotide provided herein comprises the nucleotide sequence of SEQ ID NO: 23. In a specific embodiment, the polynucleotide provided herein comprises the nucleotide sequence of SEQ ID NO: 24. In a specific embodiment, the polynucleotide provided herein comprises the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24. In a specific embodiment, the combination of polynucleotides provided herein comprises a first polynucleotide comprising the nucleotide sequence of SEQ ID NO: 23 and a second polynucleotide comprising the nucleotide sequence of SEQ ID NO: 24.

[0222] Also provided herein are polynucleotides encoding anti-KIT antibodies or fragments thereof that are optimized, for example, by codon / RNA optimization, exchange with heterologous signal sequences, and removal of mRNA instability elements. Methods for generating optimized nucleic acids encoding anti-KIT antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and / or removing inhibitory regions in mRNA can be performed by correspondingly adapting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) in the RNA can be mutated without modifying the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. Modifications can, for example, take advantage of the degeneracy of the genetic code, which uses alternative codons for the same amino acids. In some embodiments, it may be desirable to modify one or more codons to encode a conservative mutation, e.g., a similar amino acid having a similar chemical structure and properties and / or function as the original amino acid. Such methods can increase expression of an anti-KIT antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, compared to expression of an anti-KIT antibody encoded by a non-optimized polynucleotide.

[0223] In certain embodiments, an optimized polynucleotide sequence encoding an anti-KIT antibody or a fragment thereof (e.g., VL domain and / or VH domain) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-KIT antibody or a fragment thereof (e.g., VL domain and / or VH domain) described herein. In a specific embodiment, an optimized nucleotide sequence encoding an anti-KIT antibody or a fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-KIT antibody or a fragment thereof described herein. In a specific embodiment, an optimized nucleotide sequence encoding an anti-KIT antibody or a fragment thereof described herein hybridizes under high stringency, medium stringency or lower stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an anti-KIT antibody or a fragment thereof described herein. Information regarding hybridization conditions has been described, see, eg, US Patent Application Publication US 2005 / 0048549 (eg, paragraphs 72-73), which is incorporated herein by reference.

[0224] In certain embodiments, the optimized polynucleotide sequences encoding the VL regions of the antibodies described herein are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to the nucleotide sequence of SEQ ID NO: 23. In certain embodiments, the optimized polynucleotide sequences encoding the VH regions of the antibodies described herein are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to the nucleotide sequence of SEQ ID NO: 24.

[0225] The polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotides can be determined by the method. The nucleotide sequences encoding the antibodies and modified versions of these antibodies described herein can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in such a way as to generate nucleic acids encoding the antibodies. Such polynucleotides encoding the antibodies can be assembled from chemically synthesized oligonucleotides (e.g., those described in Kutmeier et al., 1994, BioTechniques 17:242), which briefly includes the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating the oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0226] Alternatively, polynucleotides encoding the antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Using such PCR amplification methods, nucleic acid comprising sequences encoding the light and / or heavy chains of the antibody can be obtained. Using such PCR amplification methods, nucleic acid comprising sequences encoding the variable light and / or variable heavy chain regions of the antibody can be obtained. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, e.g., to generate chimeric and humanized antibodies.

[0227] If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., any tissue or cell expressing the antibody, e.g., an antibody cDNA library or a cDNA library generated from hybridoma cells selected to express an antibody described herein, or nucleic acid isolated from the cells, preferably polyA+RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning, e.g., using oligonucleotide probes specific for the particular gene sequence to identify a cDNA clone from a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0228] DNA encoding the anti-KIT antibody described herein can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the anti-KIT antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from CHO GS System™ (Lonza)), or myeloma cells, to obtain the synthesis of the anti-KIT antibody in the recombinant host cells.

[0229] To generate a complete antibody, the VH or VL sequence can be amplified in a scFv clone using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and a flanking sequence to protect the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as human gamma 1 or gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as human kappa or lambda constant region. In one embodiment, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector that expresses the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, such as IgG.

[0230] The DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0231] 5.3 Host Cells and Recombinant Expression of Antibodies In certain aspects, provided herein are host cells that recombinantly express the antibodies (or antigen-binding fragments thereof) and associated expression vectors described herein. Provided herein are vectors and combinations of vectors (e.g., expression vectors) that include a polynucleotide that includes a nucleotide sequence encoding an anti-KIT antibody or antigen-binding fragment for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein are host cells that include such vectors or combinations of vectors for recombinant expression of an anti-KIT antibody (e.g., a human or humanized antibody) described herein.

[0232] Recombinant expression of an antibody described herein that immunospecifically binds to the KIT antigen (e.g., a full-length antibody, a heavy and / or light chain of the antibody, or a single-chain antibody described herein) involves the construction of an expression vector(s) containing a polynucleotide(s) encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, an antibody heavy and / or light chain, or a fragment thereof (preferably, but not necessarily, containing a heavy and / or light chain variable domain) is obtained, a vector(s) for the production of the antibody molecule can be made by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody (or VH / VL or heavy / light chain). Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the antibody and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vitro genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, an antibody heavy or light chain variable domain or fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can include, for example, the constant region of an antibody molecule (see, e.g., International Publications WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and an antibody variable domain can be cloned into such a vector for expression of an entire heavy chain, an entire light chain, or both the entire heavy and light chains.

[0233] In a specific embodiment, provided herein is a vector comprising a polynucleotide encoding the VH of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein is a vector comprising a polynucleotide encoding the VL of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein is a vector comprising a polynucleotide encoding the VH and VL of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein is a vector comprising a first polynucleotide encoding the VH of an antibody or antigen-binding fragment thereof described herein, and a second polynucleotide encoding the VL of the antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein is a combination of two vectors, the first vector of the combination comprising a first polynucleotide encoding the VH of an antibody or antigen-binding fragment thereof described herein, and the second vector of the combination comprising a second polynucleotide encoding the VL of the antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein is a vector comprising a polynucleotide encoding the heavy chain of an antibody described herein. In a specific embodiment, provided herein is a vector comprising a polynucleotide encoding the light chain of an antibody described herein. In a specific embodiment, provided herein is a vector comprising a polynucleotide encoding the heavy and light chains of the antibody described herein.In a specific embodiment, provided herein is a vector comprising a first polynucleotide encoding the VH of the antibody described herein and a second polynucleotide encoding the VL of the antibody.In a specific embodiment, provided herein is a combination of two vectors, the first vector of the combination comprising a first polynucleotide encoding the VH of the antibody described herein, and the second vector of the combination comprising a second polynucleotide encoding the VL of the antibody.

[0234] An expression vector or combination of expression vectors can be introduced into a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antibody or fragment thereof as described herein. Thus, provided herein is a host cell containing a polynucleotide or combination of polynucleotides encoding an antibody or fragment thereof as described herein, or a heavy or light chain or fragment thereof, or a single chain antibody as described herein, operably linked to a promoter for expression of such sequences in the host cell. In certain embodiments, for expression of a double chain antibody, vectors encoding both the heavy and light chains separately can be co-expressed in a host cell for expression of the entire immunoglobulin molecule, as described in more detail below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding both the heavy and light chains (or both the VH and VL) of an antibody as described herein, or a fragment thereof. In a specific embodiment, the host cell contains two different vectors, where a first vector comprises a polynucleotide encoding the heavy chain (or VH) or fragment thereof of an antibody as described herein, and a second vector comprises a polynucleotide encoding the light chain (or VL) or fragment thereof of an antibody as described herein. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain (or VH) or fragment thereof of an antibody described herein, and the second host cell comprises a second vector comprising a polynucleotide encoding the light chain (or VL) or fragment thereof of an antibody described herein.

[0235] A variety of host-expression vector systems can be used to express the antibody molecules described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems are vehicles in which coding sequences of interest can be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antibody molecules described herein in situ. These include microorganisms such as bacteria (e.g., Escherichia coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS, CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a vaccinia virus 7.5K promoter). In a specific embodiment, the cells for expression of the antibodies or antigen-binding fragments thereof described herein are CHO cells, e.g., CHO cells derived from CHO GS System™ (Lonza). In a specific embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3.Preferably, bacterial cells, such as E. coli, and more preferably, eukaryotic cells, especially for the expression of complete recombinant antibody molecules, are used for the expression of recombinant antibody molecules. Mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2). In some embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of the nucleotide sequence encoding the antibodies described herein that immunospecifically bind to the KIT antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0236] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use for the antibody molecule expressed. For example, if large quantities of such antibodies are to be produced, vectors directing the expression of high levels of fusion protein products that are easily purified for the generation of pharmaceutical compositions of the antibody molecule may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791); pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509); in which antibody coding sequences can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein. pGEX vectors can also be used to express heterologous polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0237] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0238] In mammalian host cells, several virus-based expression systems are available. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions El or E3) results in recombinant viruses that are viable in infected hosts and capable of expressing antibody molecules (see, e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0239] Furthermore, a host cell strain can be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be chosen to ensure the correct modification and processing of the expressed heterologous protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In certain embodiments, the humanized monoclonal anti-KIT antibodies described herein are produced in mammalian cells, e.g., CHO cells.

[0240] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. After introduction of the heterologous DNA, engineered cells can be allowed to grow in rich medium for 1-2 days and then switched to selection medium. The selection marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be further cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibody molecules. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with the antibody molecule.

[0241] Several selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., 1977, Cell 11:223), the hypoxanthine guanine phosphoribosyltransferase gene (Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and the adenine phosphoribosyltransferase gene (Lowy et al., 1980, Cell 22:8-17), which can be utilized in tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance has been conferred by the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215; and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).The desired recombinant clones can be selected by routine application of methods generally known in the art of recombinant DNA technology, such as those described, for example, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13 of Dracopoli et al., eds., Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties.

[0242] The expression level of antibody molecules can be increased by vector amplification (for a review see Bebbington and Hentschel, The use of vector based on gene amplification for the expression of cloned genes in mammalian cells, Vol. 3, Academic Press, New York, 1987). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0243] A host cell can be co-transfected with two or more of the expression vectors described herein, where a first vector encodes a heavy chain derived polypeptide and a second vector encodes a light chain derived polypeptide. The two vectors can contain identical selection markers that allow for equal expression of heavy and light chain polypeptides. A host cell can be co-transfected with different amounts of two or more expression vectors. For example, a host cell can be transfected with any one of the following ratios of the first expression vector and the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0244] Alternatively, a single vector can be used that can encode and express both heavy and light chain polypeptides. In such circumstances, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. Polycistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes per nucleotide sequence, or in the range of 2-5, 5-10, or 10-20 genes. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein). In such an expression vector, transcription of both genes can be driven by a promoter, but translation of mRNA from the first gene can be by a cap-dependent scanning mechanism and translation of mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.

[0245] Once an antibody molecule described herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Further, the antibodies described herein can be fused to heterologous polypeptide sequences, as described herein or otherwise known in the art to facilitate purification.

[0246] In specific embodiments, the antibodies described herein are isolated or purified. Typically, an isolated antibody is an antibody that is substantially free of other antibodies having antigen specificity different from that of the isolated antibody. For example, in certain embodiments, preparations of antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of antibodies, such as different post-translationally modified forms of antibodies or other different types of antibodies (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation.When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein.Thus, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest.In a specific embodiment, the antibody described herein is isolated or purified.

[0247] 5.4 Antibody Production The antibodies (e.g., human or humanized antibodies) described herein that immunospecifically bind to the KIT antigen (or antigen-binding fragments thereof) can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques. In certain aspects, provided herein are methods of making the antibodies described herein, comprising culturing a host cell described herein and / or expressing such an antibody using the host cell, which optionally further comprises purifying the antibody obtained from the host cell. The methods described herein utilize, unless otherwise indicated, conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields, which techniques are within the capabilities of one of ordinary skill in the art. These techniques are described in the references cited herein and are fully explained in the literature.See, e.g., Maniatis et al. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually revised); Current Protocols in Immunology, John Wiley & Sons (1987 and annually revised); Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; See Eckstein, ed. (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al., eds. (1999), Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0248] For example, humanized antibodies can be produced using a variety of techniques known in the art, including CDR grafting (EP 239,400; WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 2002, 114:117-118). 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), as well as those described, for example, in U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Publication US 2005 / 0042664 A1 (February 24, 2005), which is incorporated herein by reference.

[0249] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including techniques known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced by recombinant technology, e.g., recombinant monoclonal antibodies can be expressed by a host cell, such as a mammalian host cell.

[0250] Methods for producing and screening specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to induce lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization (e.g., the extracellular domain of human KIT). Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Additionally, animals can be immunized using RIMMS (repetitive immunization multiple sites) technology (Kilptrack et al., 1997 Hybridoma 16:381-9, incorporated herein by reference).

[0251] Non-limiting examples of myeloma cell lines include mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, Md., USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The antibodies described herein include antibody fragments that recognize specific KIT antigens and can be generated by any technique known to those of skill in the art, such as Fab and F(ab') fragments described herein. 2 Fragments may be purified using papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 F(ab') fragments can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as Fab' (Fab') and Fab' (Fab')'s. Fab fragments correspond to one of the two identical arms of an antibody molecule and contain an intact light chain paired with the VH and CHI domains of the heavy chain. 2 The fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

[0252] In one embodiment, to generate a complete antibody, a VH or VL sequence can be amplified from a template, e.g., an scFv clone, using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and a flanking sequence to protect the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, e.g., an IgG, using techniques known to those skilled in the art.

[0253] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods well known in the art. See Riechmann et al., 1999, J. Immunol. 231:25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302; U.S. Patent No. 6,005,079; and International Publication Nos. WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301.

[0254] 5.5 Treatment Methods and Medical Uses Provided herein are methods for preventing, preventing, preventing, treating, and / or managing a KIT-associated disorder or disease. Such methods include administering to a subject in need thereof a therapeutically effective amount of an anti-KIT antibody (e.g., a humanized antibody), an antigen-binding fragment thereof, a conjugate thereof, or a pharmaceutical composition described herein. In certain embodiments, also provided herein are methods for preventing, preventing, preventing, treating, or managing a KIT-associated disorder or disease.

[0255] In a specific embodiment, the methods described herein for treating a KIT-related disorder or disease provide for a reduction or amelioration of the progression, severity, and / or duration of a KIT-related disorder or disease resulting from administration of one or more therapies (including, but not limited to, administration of one or more prophylactic or therapeutic agents, e.g., an anti-KIT antibody described herein). In a further specific embodiment, the methods described herein for treating a KIT-related disorder or disease relate to alleviating one or more symptoms of a KIT-related disorder or disease. In a specific embodiment, the antibody described herein, or an antigen-binding fragment thereof, or a conjugate thereof, or the pharmaceutical composition described herein, is for use in the prevention, treatment, or management of a KIT-related disorder. In a particular embodiment, the KIT-related disease or disorder treated or managed or prevented with the anti-KIT antibody described herein, or an antigen-binding fragment thereof, or a conjugate thereof, or the pharmaceutical composition described herein, is associated with KIT expression and / or activity, e.g., involves cells that express KIT and / or exhibit KIT activity, but is not caused by or is a consequence of KIT expression or activity.

[0256] In a specific embodiment, the antibody used in the methods described herein is internalized by the cells to which it binds.In a particular embodiment, a conjugate is used in the methods described herein, wherein the conjugate comprises an antibody described herein (e.g., a humanized anti-KIT antibody), or a KIT-binding fragment thereof.In a specific embodiment, the conjugate comprises an antibody described herein (e.g., a humanized anti-KIT antibody), or a KIT-binding fragment thereof, covalently or non-covalently linked to a therapeutic agent, such as a toxin.In some embodiments, the conjugate used in the methods described herein is internalized by the cells to which it binds.

[0257] In some embodiments, KIT is abnormally (e.g., highly) expressed by cells, e.g., KIT is overexpressed. In certain embodiments, KIT expression (e.g., on the cell surface) is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than KIT expression on the surface of control cells (e.g., cells expressing normal levels of KIT, e.g., normal, e.g., human, mast cells, stem cells, brain cells, melanoblasts, or ovarian cells). In certain embodiments, KIT expression results in cell surface KIT expression at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the average KIT expression on the surface of a control cell population (e.g., a cell population expressing normal levels of KIT, e.g., a normal, e.g., human, mast cell population, stem cell population, brain cell population, melanoblast cell population, or ovarian cell population). In specific embodiments, such control cells can be obtained from or derived from a healthy individual (e.g., a healthy human). In some embodiments, KIT can be abnormally upregulated in a particular cell type, regardless of whether KIT is abnormally expressed on the cell surface. In certain embodiments, KIT signaling or activity can be abnormally upregulated in a particular cell type, regardless of whether KIT is abnormally expressed on the cell surface. In certain embodiments, the KIT signaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the KIT signaling of a control cell (e.g., a cell comprising normal KIT signaling, e.g., a mast cell, a stem cell, a brain cell, a melanoblast, or an ovarian cell). In certain embodiments, the KIT signaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the average KIT signaling of a control cell population (e.g., a cell population exhibiting normal KIT signaling, e.g., a normal, e.g., human, mast cell population, a stem cell population, a brain cell population, a melanoblast population, or an ovarian cell population). In certain embodiments, the normal, abnormal, or excessive cell signaling is caused by binding of KIT to a KIT ligand.In other embodiments, the aberrant or excessive cell signaling occurs independently of binding of KIT to a KIT ligand.

[0258] In some embodiments, KIT-related disorders or diseases may be characterized by gain-of-function KIT activity, increased KIT activity, or overexpression of KIT. In one embodiment, KIT-related disorders or diseases are caused or result from gain-of-function KIT activity or expression, e.g., overexpression, completely or partially. In some embodiments, gain-of-function KIT activity may occur independently of KIT ligand (e.g., SCF) binding to KIT receptor. In certain embodiments, high expression or overexpression of KIT in a cell refers to an expression level that is at least about 35%, 45%, 55%, or 65% greater than the expression level of a reference cell known to have normal KIT expression or KIT activity, or the average expression level of KIT in a population of cells or samples known to have normal KIT expression or KIT activity. The expression level of KIT can be assessed by methods described herein or known to those skilled in the art (e.g., Western blotting or immunohistochemistry). In certain embodiments, KIT-related disorder or disease is characterized by KIT activity that is higher than normal and contributes to cell transformation, neoplasia, and tumorigenesis.In certain embodiments, high KIT activity or increased KIT activity in cells refers to KIT activity level that is at least about 35%, 45%, 55%, or 65% higher than the expression level of reference cells known to have normal KIT activity, or the average level of KIT activity in a population of cells or samples known to have normal KIT activity.Non-limiting examples of KIT activity include tyrosine phosphorylation of the cytoplasmic domain of KIT, and downstream signal transduction of KIT, such as Stat or Akt signal transduction.

[0259] In some embodiments, the KIT-related disorder is a mast cell-related disorder, an eosinophil-related disorder, a cancer, asthma, an inflammatory disease, rheumatoid arthritis, an allergic inflammation, an inflammatory bowel disease, a gastrointestinal disorder, or a fibrosis. In some embodiments, the KIT-related disorder is a fibrosis or an inflammatory disorder, for example, an inflammatory bowel disease (IBD), for example, Crohn's disease (CD) or ulcerative colitis (UC). In other embodiments, the KIT-related disorder is a cancer, for example, a lung cancer (for example, small cell lung cancer), a leukemia, a neuroblastoma, a melanoma, a sarcoma (for example, Ewing's sarcoma), or a gastrointestinal stromal tumor (GIST). In other embodiments, the KIT-related disorder is a systemic mast cell disorder (for example, mastocytosis), a blood disorder, a fibrosis (for example, idiopathic pulmonary fibrosis (TPF), scleroderma, or myelofibrosis), or an inflammatory disease, for example, asthma, rheumatoid arthritis, an inflammatory bowel disease, or an allergic inflammation.

[0260] In a specific embodiment, the KIT-associated disorder is a mast cell-associated disorder. In a specific embodiment, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

[0261] Mast cells, derived from bone marrow precursor cells, are found in connective tissues throughout the body and are the most abundant giant cells in the submucosa and dermis. They contain large granules that store a variety of mediator molecules, including the vasoactive amine histamine, and have the high affinity Fes receptor (FcsRI) that allows them to bind IgE monomers. Antigen binding to mast cell-bound IgE induces mast cell degranulation and activation, resulting in local or systemic immediate hypersensitivity reactions. Thus, mast cells play an important role in inflammatory and allergic responses. However, without proper balance and regulation, mast cells can also be responsible for harmful over-reactions to antigens observed in disorders such as anaphylaxis, atopy, and rhinitis.

[0262] KIT signaling is important for mast cell development and homeostasis, e.g., proliferation of mast cells from their precursor cells and their subsequent maturation and survival in resident tissues, homing of mast cells to their retention sites in vivo, and promoting adhesion of mast cells to extracellular matrix proteins. Activating mutations in KIT, such as at amino acid residues 816 or 560 of KIT, are associated with mastocytosis, a condition characterized by overproduction of mast cells and gastrointestinal stromal cell tumors (GISTs).

[0263] In certain embodiments, the methods described herein for preventing, treating, or managing a KIT-mediated disorder, e.g., a fibrotic or inflammatory disease (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation) in a subject in need thereof, comprise administering a therapeutically effective amount of an anti-KIT antibody described herein to the subject, the method comprising: (i) reducing or preventing the severity of a fibrotic or inflammatory disease (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation) and / or one or more symptoms associated therewith. (ii) reducing the duration of one or more symptoms associated with fibrosis or inflammatory disease (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (iii) preventing recurrence of fibrosis or inflammatory disease (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation), (iv) reducing hospitalization of the subject; (v) reducing the length of hospitalization; (vi) reducing the duration of fibrosis or inflammatory disease (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation) and / or one or more symptoms associated therewith. (vii) enhancing or improving the therapeutic efficacy of another therapy (e.g., an anti-inflammatory therapy such as a steroid); (viii) increasing the number of patients in remission (i.e., a period characterized by the absence or minimal symptoms associated with an inflammatory disease); (ix) increasing the length of time that patients are in remission; (x) reducing hospitalization rates; (xi) reducing the number of symptoms associated with a fibrotic or inflammatory disease (e.g., asthma, chronic rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (xii) reducing the concentration of one or more inflammatory mediators (e.g., cytokines or interleukins) in a biological sample (e.g., plasma, serum, cerebrospinal fluid, urine, or any other biological fluid) of a subject having a fibrotic or inflammatory disease (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); and (xiii) improving quality of life as assessed by methods well known in the art, e.g., questionnaires.

[0264] Other non-limiting examples of KIT-related disorders or diseases include systemic mast cell disorders (e.g., mastocytosis), blood disorders, fibrosis (e.g., idiopathic pulmonary fibrosis (TPF), scleroderma, or myelofibrosis).

[0265] As used herein, the term "mast cell-associated disorder" or "mast cell-associated disorders" refers to disorders in which mast cell activity is involved in the pathology and / or mast cells are found in abnormal amounts, e.g., above or below normal amounts. For example, a mast cell-associated disorder can potentially manifest as an accumulation of pathological mast cells in any or all organs and tissues and / or an abnormal release of one or more mast cell mediators, such as inflammatory mediators. Non-limiting examples of inflammatory mediators released by mast cells include any of: (i) granule-associated mediators, including histamine, serotonin (5-hydroxytryptamine), and various proteases and peptidases; (ii) eicosanoids, such as prostaglandin D2 (PGD2) and leukotriene C4 (LTC4); and (iii) cytokines, including interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor a (TNFa), and chemokines, including CCL-2, CCL-3, CCL-5, and CXCL8.

[0266] In a specific embodiment, the mast cell-associated disorder is a mast cell-associated disorder of the nervous system, e.g., the central nervous system, e.g., NMO, NMOSD, MS, or NF (e.g., NF type 1 (NF1), NF type 2 (NF2), or schwannomatosis).

[0267] MS is a chronic inflammatory demyelinating disorder of the central nervous system (brain and spinal cord) involving episodes in which white matter in the brain or spinal cord becomes inflamed and then damaged by the individual's own immune system. These inflamed areas become scarred in the brain and spinal cord. The damage destroys the ability of parts of the nervous system to communicate, resulting in a variety of symptoms including physical, mental, and / or psychiatric problems. Forms of MS include, but are not limited to, relapsing, where symptoms occur in a single attack, and progressive, where symptoms accumulate over time. Guidelines for diagnosing MS are described, see, e.g., National Collaborating Centre for Chronic Conditions (UK), "Multiple Sclerosis: National Clinical Guideline for Diagnosis and Management in Primary and Secondary Care," London: Royal College of Physicians (UK), 2004, (NICE Clinical Guideline, No. 8.), available at http: / / www.ncbi.nlm.nih.gov / books / NBK48919 / . Symptoms of MS may manifest as any neurological symptom or sign, such as autonomic, visual, motor, and sensory problems. Non-limiting examples of symptoms of MS include loss of sensitivity or changes in sensation, such as pins and needles or numbness, muscle weakness, very noticeable reflexes, muscle spasms or difficulty moving, impaired coordination and balance (ataxia), problems with speaking or swallowing, visual problems (nystagmus, optic neuritis or diplopia), fatigue, acute or chronic pain, bladder and bowel disorders, emotional problems such as depression or unstable mood, Uthof's phenomenon (worsening symptoms with exposure to higher than normal temperatures), and Lhermitte's sign (an electrical sensation down the back when bending the neck).In a specific aspect, provided herein is a method of preventing, treating, alleviating, or managing one or more of these symptoms of MS by administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to KIT (e.g., human KIT).

[0268] Other non-limiting examples of mast cell-related disorders include, for example, anaphylaxis, atopic diseases, mast cell activation syndrome, allergic rhinitis, food and venom-related allergies (e.g., nut, shellfish, fish, hymenoptera venom, or bee sting allergies), psoriasis, atopic dermatitis, rosacea, eczema, tubulointerstitial nephritis, glomerulonephritis, diabetic nephropathy, allograft rejection, amyloidosis, renal vascular ischemia, reflux nephropathy, polycystic kidney disease, drug-induced nephropathy, post-transplant ion fibrosis, and liver fibrosis (e.g., alcohol consumption, viral hepatitis B and C, and non-alcoholic steatohepatitis (NASH)), parasitic infections (e.g., schistosomiasis, amebiasis, echinococcosis), and non-IgE mast cell-mediated activation, for example, angioedema and anaphylaxis.

[0269] Alternatively, the mast cell-related disorder may be urticaria, particularly chronic spontaneous urticaria, chronic idiopathic urticaria, and chronic induced urticaria (i.e., chronic inducible urticaria). The mast cell-related disorder may be chronic urticaria, including chronic urticaria (CIndU). In a specific embodiment, the mast cell-related disorder is chronic induced urticaria. Chronic induced urticaria is a form of urticaria that has an associated trigger, usually resulting in wheals (hives) or angioedema. In a specific embodiment, the chronic induced urticaria is cold urticaria (ColdU). People suffering from cold urticaria experience symptoms such as itching, burning wheals and angioedema when the skin is exposed to temperatures lower than the skin temperature. In another specific embodiment, the chronic induced urticaria is symptomatic dermographism (SD). Symptomatic dermographism is characterized by the development of a wheal and flare response in response to stroking, scratching or rubbing the skin, usually occurring within minutes of the triggering stimulus. In another specific embodiment, the chronic induced urticaria is cholinergic urticaria. Cholinergic urticaria is a type of urticaria that is triggered by the body's sweating response to active or passive body temperature elevation and is characterized by a small (1-4 mm) wheal surrounded by a bright red flare. Common triggers include exercise, hot baths / showers, fever, occlusive dressings, ingestion of spicy foods, and mental stress. In another specific embodiment, the chronically induced urticaria is heat urticaria. In another specific embodiment, the chronically induced urticaria is delayed onset pressure ulcers. In another specific embodiment, the chronically induced urticaria is solar urticaria. In another specific embodiment, the chronically induced urticaria is vibratory urticaria. In another specific embodiment, the chronically induced urticaria is contact urticaria. In another specific embodiment, the chronically induced urticaria is aqueous urticaria. Antihistamines are approved therapeutic agents for chronically induced urticaria.

[0270] In various embodiments, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard of care therapies for the disorder. In certain embodiments, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed antihistamine treatment(s) for the disorder. In a specific embodiment, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed H1 antihistamine treatment(s) for the disorder. In a specific embodiment, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed H2-antihistamine treatment(s) for the disorder. In a specific embodiment, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed both H1- and H2-antihistamine treatment(s) for the disorder. In some embodiments, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed one or more leukotriene receptor antagonist treatment(s) for the disorder. In certain embodiments, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed treatment(s) with one or more immunomodulatory or anti-inflammatory agents for the disorder. In a specific embodiment, a patient with a mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed treatment with an anti-IgE antibody, e.g., an IgE inhibitor, such as omalizumab (Xolair®) or ligelizumab, for the disorder.In a specific embodiment, a patient with a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis has failed treatment with an anti-IL-4R antibody for the disorder, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®). In a specific embodiment, a patient with a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis has failed treatment with an anti-IL-5R antibody for the disorder, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®). In a specific embodiment, a patient with a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis has failed treatment with an anti-IL-5 antibody for the disorder, e.g., an IL-5 inhibitor such as mepolizumab. In a specific embodiment, a patient with a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis has failed treatment with an anti-Siglec 8 antibody, e.g., a Siglec 8 inhibitor, such as lirentelimab, for the disorder. In a specific embodiment, a patient with a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis has failed treatment with an anti-TSLP antibody or an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor, such as tezepelumab (Tezspire™), for the disorder. In a specific embodiment, a patient with a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis has failed treatment with an anti-C5aR antibody, e.g., a C5aR inhibitor, such as abdoralimab, for the disorder. In a specific embodiment, a patient having a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, has failed treatment with an anti-CD200R antibody, e.g., a CD200R inhibitor, such as LY3454738, for the disorder.In certain embodiments, a patient having a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed treatment(s) with one or more Bruton's tyrosine kinase (BTK) inhibitors for the disorder, e.g., remibrutinib and / or rilzabrutinib. In certain embodiments, a patient having a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment(s) with one or more leukotriene receptor antagonists, (3) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec antibody, or ... and / or (3) have failed treatment(s) with one or more BTK inhibitors, such as remibrutinib and / or rilzabrutinib; (4) have failed treatment(s) with one or more anti-CD200R antibodies, such as a CD200R inhibitor such as LY3454738; or (5) have failed treatment(s) with one or more anti-CD200R antibodies, such as a CD200R inhibitor such as LY3454738;In certain embodiments, a patient having a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)) for the disorder, and (2) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec 8 antibody, e.g., an anti-Siglec 9 antibody, e.g., an anti-Siglec 10 antibody, e.g., an anti-Siglec 12 antibody, e.g., an anti-Siglec 14 antibody, e.g., an anti-Siglec 15 antibody, e.g., an anti-Siglec 16 antibody, e.g., an anti-Siglec 17 antibody, e.g., an anti-Siglec 18 antibody, e.g., an anti-Siglec 19 antibody, e.g., an anti-Siglec 20 antibody, e.g., an anti-Siglec 21 antibody, e.g., an anti-Siglec 22 antibody, e.g., an anti-Siglec 23 antibody, have failed treatment(s) with an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™), an anti-C5aR antibody, e.g., a C5aR inhibitor such as abdoralimab, and / or an anti-CD200R antibody, e.g., a CD200R inhibitor such as LY3454738. In certain embodiments, a patient having a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (3) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®), for the disorder. In certain embodiments, a patient having a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed treatment with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)) for the disorder, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab.In certain embodiments, a patient having a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed treatment with (1) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®), for the disorder.

[0271] A patient is considered to have failed treatment if the disorder is refractory to treatment, resistant to treatment, recurs after treatment, and / or if the patient discontinues treatment due to intolerance to the treatment.

[0272] In various embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is refractory to one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard of care therapies for the disorder. In certain embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is refractory to antihistamine treatment(s). In a specific embodiment, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is refractory to H1-antihistamine treatment(s). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to H2-antihistamine treatment(s). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to both H1- and H2-antihistamine treatment. In certain embodiments, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment(s) with one or more leukotriene receptor antagonists. In certain embodiments, mast cell-related disorders such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorders such as eosinophilic esophagitis are refractory to treatment(s) with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, mast cell-related disorders such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorders such as eosinophilic esophagitis are refractory to treatment with an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab. In a specific embodiment, mast cell-related disorders such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorders such as eosinophilic esophagitis are refractory to treatment with an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®).In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment with an anti-IL-5R antibody, e.g., an IL-5R inhibitor, such as benralizumab (Fasenra®). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment with an anti-IL-5 antibody, e.g., an IL-5 inhibitor, such as mepolizumab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment with an anti-Siglec 8 antibody, e.g., a Siglec 8 inhibitor, such as lirentelimab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment with an anti-TSLP antibody or an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor, such as tezepelumab (Tezspire™). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment with an anti-C5aR antibody, e.g., a C5aR inhibitor, such as abdoralimab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is refractory to treatment with an anti-CD200R antibody, e.g., a CD200R inhibitor, such as LY3454738. In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is refractory to treatment(s) with one or more BTK inhibitors, e.g., remibrutinib and / or rilzabrutinib.In a specific embodiment, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment(s) with one or more leukotriene receptor antagonists, (3) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec antibody, or an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-IL-5 antibody, or an anti- ... and / or (3) the patient is refractory to treatment(s) with one or more BTK inhibitors, such as remibrutinib and / or rilzabrutinib; (4) an anti-TSLP antibody or an anti-TSLPR antibody, such as a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™); an anti-C5aR antibody, such as a C5aR inhibitor such as abdoralimab; and / or an anti-CD200R antibody, such as a CD200R inhibitor such as LY3454738; and / or (5) a BTK inhibitor, such as remibrutinib and / or rilzabrutinib.In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec 8 antibody, e.g., an anti-Siglec 9 antibody, e.g., an anti-Siglec 10 antibody, e.g., an anti-Siglec 12 antibody, e.g., an anti-Siglec 14 antibody, e.g., an anti-Siglec 15 antibody, e.g., an anti-Siglec 16 antibody, e.g., an anti-Siglec 17 antibody, e.g., an anti-Siglec 18 antibody, e.g., an anti-Siglec 19 antibody, e.g., an anti-Siglec 20 antibody, e.g., an anti-Siglec 21 antibody, e.g., an anti-Siglec 22 antibody, e.g., an anti-Siglec 23 antibody, e.g., an anti-Siglec 24 antibody, e.g., an anti-Siglec 25 antibody, e.g., an anti- The patient is refractory to treatment(s) with an anti-TSLPR antibody, an anti-TSLP antibody or an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™), an anti-C5aR antibody, e.g., a C5aR inhibitor such as abdoralimab, and / or an anti-CD200R antibody, e.g., a CD200R inhibitor such as LY3454738. In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is refractory to treatment with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory to (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab.In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory to treatment with (1) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®).

[0273] In various embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is resistant to one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard of care therapies for the disorder. In certain embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is resistant to antihistamine treatment(s). In a specific embodiment, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is resistant to H1-antihistamine treatment(s). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to H2-antihistamine treatment(s). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to both H1- and H2-antihistamine treatment. In certain embodiments, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment(s) with one or more leukotriene receptor antagonists. In certain embodiments, mast cell-related disorders such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorders such as eosinophilic esophagitis are resistant to treatment(s) with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, mast cell-related disorders such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorders such as eosinophilic esophagitis are resistant to treatment with an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab. In a specific embodiment, mast cell-related disorders such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorders such as eosinophilic esophagitis are resistant to treatment with an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®).In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment with an anti-IL-5R antibody, e.g., an IL-5R inhibitor, such as benralizumab (Fasenra®). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment with an anti-IL-5 antibody, e.g., an IL-5 inhibitor, such as mepolizumab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment with an anti-Siglec 8 antibody, e.g., a Siglec 8 inhibitor, such as lirentelimab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment with an anti-TSLP antibody or an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor, such as tezepelumab (Tezspire™). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment with an anti-C5aR antibody, e.g., a C5aR inhibitor, such as abdoralimab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is resistant to treatment with an anti-CD200R antibody, e.g., a CD200R inhibitor, such as LY3454738. In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is refractory to treatment with one or more BTK inhibitors, e.g., remibrutinib and / or rilzabrutinib.In a specific embodiment, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment(s) with one or more leukotriene receptor antagonists, (3) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec antibody, or an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-IL-5 antibody, or an anti- ... and / or (3) the patient is resistant to treatment(s) with one or more BTK inhibitors, such as remibrutinib and / or rilzabrutinib; (4) an anti-TSLP antibody or an anti-TSLPR antibody, such as a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™); an anti-C5aR antibody, such as a C5aR inhibitor such as abdoralimab; and / or an anti-CD200R antibody, such as a CD200R inhibitor such as LY3454738; and / or (5) a BTK inhibitor, such as remibrutinib and / or rilzabrutinib.In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec 8 antibody, e.g., an anti-Siglec 9 antibody, e.g., an anti-Siglec 10 antibody, e.g., an anti-Siglec 12 antibody, e.g., an anti-Siglec 14 antibody, e.g., an anti-Siglec 15 antibody, e.g., an anti-Siglec 16 antibody, e.g., an anti-Siglec 17 antibody, e.g., an anti-Siglec 18 antibody, e.g., an anti-Siglec 19 antibody, e.g., an anti-Siglec 20 antibody, e.g., an anti-Siglec 21 antibody, e.g., an anti-Siglec 22 antibody, e.g., an anti-Siglec 23 antibody, e.g., an anti-Siglec 24 antibody, e.g., an anti-Siglec 25 antibody, e.g., an anti- The patient is resistant to treatment(s) with an anti-TSLPR antibody, an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™), an anti-C5aR antibody, e.g., a C5aR inhibitor such as abdoralimab, and / or an anti-CD200R antibody, e.g., a CD200R inhibitor such as LY3454738. In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is refractory to treatment with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)); (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab; and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory to (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab.In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is refractory to (1) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (2) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®).

[0274] In various embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or the eosinophil-related disorder, such as eosinophilic esophagitis, is both refractory and resistant to one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard of care therapies for the disorder. In certain embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or the eosinophil-related disorder, such as eosinophilic esophagitis, is both refractory and resistant to antihistamine treatment(s). In a specific embodiment, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or the eosinophil-related disorder, such as eosinophilic esophagitis, is both refractory and resistant to H1-antihistamine treatment(s). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is both refractory and resistant to H2-antihistamine treatment(s). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is both refractory and resistant to both H1- and H2-antihistamine treatment. In certain embodiments, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is both refractory and resistant to treatment(s) with one or more leukotriene receptor antagonists. In certain embodiments, a mast cell-associated disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment(s) with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, a mast cell-associated disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an IgE inhibitor such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab.In a specific embodiment, a mast cell-associated disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®). In a specific embodiment, a mast cell-associated disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®). In a specific embodiment, a mast cell-associated disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an anti-Siglec 8 antibody, e.g., a Siglec 8 inhibitor, such as lirentelimab. In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an anti-TSLP antibody or an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor, such as tezepelumab (Tezspire™). In a specific embodiment, a mast cell-related disorder such as urticaria (e.g., chronically triggered urticaria) or an eosinophil-related disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with an anti-C5aR antibody, e.g., a C5aR inhibitor, such as abdoralimab. In a specific embodiment, a mast cell-associated disorder such as urticaria (e.g., chronically-induced urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment with a CD200R inhibitor such as an anti-CD200R antibody, e.g., LY3454738. In certain embodiments, a mast cell-associated disorder such as urticaria (e.g., chronically-induced urticaria) or an eosinophil-associated disorder such as eosinophilic esophagitis is both refractory and resistant to treatment(s) with one or more BTK inhibitors, e.g., remibrutinib and / or rilzabrutinib.In a specific embodiment, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment(s) with one or more leukotriene receptor antagonists, (3) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec antibody, or an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-IL-5 antibody, or an anti- ... and / or (3) are both refractory and resistant to treatment(s) with one or more BTK inhibitors, e.g., Siglec 8 inhibitors such as lirentelimab, anti-TSLP or anti-TSLPR antibodies, e.g., TSLP or TSLPR inhibitors such as tezepelumab (Tezspire™), anti-C5aR antibodies, e.g., C5aR inhibitors such as abdoralimab, and / or anti-CD200R antibodies, e.g., CD200R inhibitors such as LY3454738); and / or (4) are refractory and resistant to treatment(s) with one or more BTK inhibitors, e.g., remibrutinib and / or rilzabrutinib.In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec 8 antibody, e.g., an anti-Siglec 9 antibody, e.g., an anti-Siglec 10 antibody, e.g., an anti-Siglec 12 antibody, e.g., an anti-Siglec 14 antibody, e.g., an anti-Siglec 15 antibody, e.g., an anti-Siglec 16 antibody, e.g., an anti-Siglec 17 antibody, e.g., an anti-Siglec 18 antibody, e.g., an anti-Siglec 19 antibody, e.g., an anti-Siglec 20 antibody, e.g., an anti-Siglec 21 antibody, e.g., an anti-Siglec 22 antibody, e.g., an anti-Siglec 23 antibody, e.g., an anti-Siglec 24 antibody, e.g., an anti-Siglec 25 antibody, e.g., an anti- In some embodiments, the disease is both refractory and resistant to treatment(s) with an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™), an anti-C5aR antibody, e.g., a C5aR inhibitor such as abdoralimab, and / or an anti-CD200R antibody, e.g., a CD200R inhibitor such as LY3454738. In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is both refractory and resistant to (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (3) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is both refractory and resistant to (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab.In certain embodiments, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is both refractory and resistant to (1) treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab, and (2) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupilumab (Dupixent®).

[0275] In various embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or the eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard of care therapies for the disorder. In certain embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or the eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after antihistamine treatment(s). In a specific embodiment, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria), or the eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after H1-antihistamine treatment(s). In a specific embodiment, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after H2-antihistamine treatment(s). In a specific embodiment, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after both H1- and H2-antihistamine treatment. In certain embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after treatment(s) with one or more leukotriene receptor antagonists. In certain embodiments, the mast cell-associated disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after treatment(s) with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, the mast cell-associated disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after treatment with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab (Xolair®) or ligelizumab.In a specific embodiment, the mast cell-associated disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after treatment with an anti-IL-4R antibody, e.g., an IL-4R inhibitor, such as dupilumab (Dupixent®). In a specific embodiment, the mast cell-associated disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after treatment with an anti-IL-5R antibody, e.g., an IL-5R inhibitor, such as benralizumab (Fasenra®). In a specific embodiment, the mast cell-associated disorder, such as urticaria (e.g., chronically triggered urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred after treatment with an anti-IL-5 antibody, e.g., an IL-5 inhibitor, such as mepolizumab. In a specific embodiment, the mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or the eosinophil-related disorder such as eosinophilic esophagitis is a recurrent disorder that recurs after treatment with an anti-Siglec 8 antibody, e.g., a Siglec 8 inhibitor such as lirentelimab. In a specific embodiment, the mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or the eosinophil-related disorder such as eosinophilic esophagitis is a recurrent disorder that recurs after treatment with an anti-TSLP antibody or an anti-TSLPR antibody, e.g., a TSLP or TSLPR inhibitor such as tezepelumab (Tezspire™). In a specific embodiment, the mast cell-related disorder such as urticaria (e.g., chronically induced urticaria) or the eosinophil-related disorder such as eosinophilic esophagitis is a recurrent disorder that recurs after treatment with an anti-C5aR antibody, e.g., a C5aR inhibitor such as abdolalimab. In a specific embodiment, the mast cell-associated disorder, such as urticaria (e.g., chronically-induced urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred following treatment with a CD200R inhibitor, such as an anti-CD200R antibody, e.g., LY3454738. In certain embodiments, the mast cell-associated disorder, such as urticaria (e.g., chronically-induced urticaria) or eosinophil-associated disorder, such as eosinophilic esophagitis, is a recurrent disorder that has recurred following treatment(s) with one or more BTK inhibitors, e.g., remibrutinib and / or rilzabrutinib.In a specific embodiment, a mast cell-associated disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-associated disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), (2) treatment(s) with one or more leukotriene receptor antagonists, (3) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec antibody, or an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-IL-5 antibody, or an anti- ... and / or (3) a treatment(s) with one or more BTK inhibitors, e.g., remibrutinib and / or rilzabrutinib.In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic inducible urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with (1) antihistamine treatment(s) (e.g., H1- and / or H2-antihistamine treatment(s)), and (2) one or more immunomodulatory or anti-inflammatory agents (e.g., an anti-IgE antibody, e.g., an IgE inhibitor such as omalizumab (Xolair®) or ligelizumab, an anti-IL-4R antibody, e.g., an IL-4R inhibitor such as dupilumab (Dupixent®), an anti-IL-5R antibody, e.g., an IL-5R inhibitor such as benralizumab (Fasenra®), an anti-IL-5 antibody, e.g., an IL-5 inhibitor such as mepolizumab, an anti-Siglec 8 antibody, e.g., an anti-Siglec 9 antibody, e.g., an anti-Siglec 10 antibody, e.g., an anti-Siglec 12 antibody, e.g., an anti-Siglec 14 antibody, e.g., an anti-Siglec 15 antibody, e.g., an anti-Siglec 16 antibody, e.g., an anti-Siglec 17 antibody, e.g., an anti-Siglec 18 antibody, e.g., an anti-Siglec 19 antibody, e.g., an anti-Siglec 20 antibody, e.g., an anti-Siglec 21 antibody, e.g., an anti-Siglec 22 antibody, e.g., an anti-Siglec 23 antibody, e.g., an anti-Siglec 24 antibody, e...

Claims

1. 1. An antibody that immunospecifically binds to human KIT, (i) a light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; (ii) a heavy chain variable region ("VH") comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and (iii) a modified human IgG1 Fc region or domain containing the non-natural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference; The antibody comprising:

2. 2. The antibody of claim 1, wherein said modified human IgG1 Fc region or domain further comprises the unnatural amino acids 252Y, 254T and 256E, as numbered by the EU index as set forth in the Kabat reference.

3. (i) the VL has the amino acid sequence: 【Chemistry 1】 (where X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid having an aliphatic or aliphatic hydroxyl side chain, and X K3 is an amino acid having an aliphatic hydroxyl side chain, and X K4 is an amino acid having an aliphatic hydroxyl side chain or is P; X K5 is an amino acid having a charged or acidic side chain, and X K6 is an amino acid having an aromatic side chain; (ii) the VH has the amino acid sequence: 【Chemistry 2】 (where X H1 is an amino acid having an aliphatic side chain, and X H2 is an amino acid having an aliphatic side chain, and X H3 is an amino acid with a polar or basic side chain, and X H4 is an amino acid having an aliphatic side chain, and X H5 is an amino acid having an aliphatic side chain, and X H6 is an amino acid with an acidic side chain, and X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 is an amino acid having an aliphatic hydroxyl side chain, The antibody of claim 1 or 2.

4. X K1 is an amino acid F or S, and X K2 is an amino acid A or S, and X K3 is the amino acid T or S, and X K4 is the amino acid S or P, and X K5 is the amino acid D or T, and X K6 is an amino acid F or Y, and X H1 is an amino acid L or V, and X H2 is an amino acid L or V, and X H3 is an amino acid K or R, and X H4 is an amino acid V or A, and X H5 is an amino acid L or I, and X H6 is amino acid E or D, and X H7 is an amino acid Q or E, and X H8 is the amino acid S or T.

5. (i) the VL comprises the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16; and (ii) The antibody of any one of claims 1 to 4, wherein the VH comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11 or 12.

6. (i) a VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified human IgG1 Fc region or domain containing the non-natural amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in the Kabat reference; The antibody of any one of claims 1 to 5, comprising:

7. (i) a VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified human IgG1 Fc region or domain comprising the non-natural amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index as set forth in the Kabat reference; The antibody of any one of claims 1 to 5, comprising:

8. Amino acid sequence: 【Chemistry 3】 The antibody of any one of claims 1 to 5, comprising a heavy chain comprising:

9. Amino acid sequence: 【Chemistry 4】 The antibody of any one of claims 1 to 5, comprising a light chain comprising:

10. Amino acid sequence: 【Chemistry 5】 and a heavy chain comprising the amino acid sequence: 【Chemistry 6】 The antibody of any one of claims 1 to 5, comprising a light chain comprising:

11. A conjugate comprising the antibody of any one of claims 1 to 10 linked to an agent.

12. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10, or a conjugate according to claim 11.

13. An antibody according to any one of claims 1 to 10, or a polynucleotide or combination of polynucleotides comprising a nucleotide sequence encoding the VH and VL of said antibody.

14. A vector or a combination of vectors comprising a polynucleotide or a combination of polynucleotides according to claim 13.

15. A host cell comprising a vector or combination of vectors according to claim 14 or a polynucleotide or combination of polynucleotides according to claim 13.

16. A kit comprising the antibody of any one of claims 1 to 10, the conjugate of claim 11, or the pharmaceutical composition of claim 12.

17. A pharmaceutical composition for preventing, treating or managing a KIT-associated disorder, comprising the antibody of any one of claims 1 to 10, the conjugate of claim 11, or the pharmaceutical composition of claim 12.

18. 18. The pharmaceutical composition of claim 17, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disease, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.

19. 19. The pharmaceutical composition of claim 18, wherein the KIT-associated disorder is a mast cell-associated disorder.

20. 20. The pharmaceutical composition of claim 19, wherein the mast cell-related disorder is chronic urticaria.

21. 21. The pharmaceutical composition of claim 20, wherein the chronic urticaria is chronic inducible urticaria.

22. 22. The pharmaceutical composition of claim 21, wherein the chronically induced urticaria is cold urticaria.

23. 22. The pharmaceutical composition of claim 21, wherein the chronically induced urticaria is symptomatic dermatographia.

24. 22. The pharmaceutical composition of claim 21, wherein the chronically triggered urticaria is cholinergic urticaria.

25. 21. The pharmaceutical composition of claim 20, wherein the chronic urticaria is chronic spontaneous urticaria.

26. The pharmaceutical composition of claim 18, wherein the KIT-associated disorder is an eosinophil-associated disorder.

27. The pharmaceutical composition of any one of claims 17 to 26, further comprising a second therapeutic agent to be administered to the subject.

28. 28. The pharmaceutical composition of claim 27, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulator, or an anti-inflammatory agent.

29. 13. An in vitro method for inhibiting KIT activity in a cell expressing KIT, comprising contacting said cell with an effective amount of an antibody according to any one of claims 1 to 10, a conjugate according to claim 11, or a pharmaceutical composition according to claim 12.

30. 30. The method of claim 29, wherein the method inhibits KIT activity by at least about 10% in cells expressing KIT.

31. An in vitro method for obtaining data for identifying a subject having a KIT-associated disorder, the method comprising contacting a cell or a sample obtained from the subject with an antibody according to any one of claims 1 to 10, and detecting the expression level of KIT in the cell or the sample.

32. 16. A method for producing an antibody, comprising culturing a host cell according to claim 15 and / or expressing said antibody using said host cell.

33. 33. The method of claim 32, further comprising purifying the antibody obtained from the host cell.

34. A pharmaceutical composition for preventing, treating, or managing chronic urticaria in a subject, comprising: (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT; (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent; or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate.

35. 35. The pharmaceutical composition of claim 34, wherein the human KIT comprises the amino acid sequence of SEQ ID NO:

1.

36. The pharmaceutical composition of claim 34 or 35, wherein the antibody specifically binds to the D4 or D5 region of human KIT.

37. The pharmaceutical composition of any one of claims 34 to 36, wherein the antibody comprises a modified Fc region or domain.

38. The pharmaceutical composition of any one of claims 34 to 37, wherein the antibody comprises a modified human Fc region or domain.

39. The pharmaceutical composition of any one of claims 34 to 38, wherein the antibody has reduced Fc receptor binding activity.

40. The pharmaceutical composition of claim 39, wherein the antibody has reduced FcγR binding activity.

41. The pharmaceutical composition according to any one of claims 34 to 40, wherein the antibody does not induce significant degranulation of human mast cells expressing FcgRI.

42. The pharmaceutical composition of any one of claims 34 to 41, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.

43. The antibody, (A)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (C)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32, respectively; (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 27, respectively; or (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; The pharmaceutical composition according to any one of claims 34 to 42, comprising:

44. 44. The pharmaceutical composition of claim 43, wherein the chronic urticaria is chronic triggered urticaria.

45. 45. The pharmaceutical composition of claim 44, wherein the chronic triggered urticaria is cold urticaria.

46. 45. The pharmaceutical composition of claim 44, wherein the chronically induced urticaria is symptomatic dermatographia.

47. 45. The pharmaceutical composition of claim 44, wherein the chronically triggered urticaria is cholinergic urticaria.

48. 44. The pharmaceutical composition of claim 43, wherein the chronic urticaria is chronic spontaneous urticaria.

49. The pharmaceutical composition of any one of claims 34 to 48, further comprising a second therapeutic agent to be administered to the subject.

50. 50. The pharmaceutical composition of claim 49, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulator, or an anti-inflammatory agent.

51. 11. A method for preventing, treating or managing a KIT-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of any one of claims 1 to 10.

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