Anti-KIT antibody formulations and methods

Abstract

Provided herein are pharmaceutical compositions comprising antibodies that immunospecifically bind to receptor tyrosine kinase KIT and uses thereof. Kits of such pharmaceutical compositions and methods for preparing such pharmaceutical compositions are also provided. KIT (or c-Kit) is a type III receptor tyrosine kinase encoded by a c-kit gene. The KIT comprises five extracellular immunoglobulin (ig)-like domains, a single transmembrane region, an inhibitory cytoplasm proximal membrane domain, and a breaking cytoplasm kinase domain separated by a kinase insertion segment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 369,644, filed on July 27, 2022, which is incorporated herein by reference in its entirety.

[0003] References to sequence listings submitted electronically

[0004] This application incorporates by reference a Sequence Listing, which is submitted with this application as an XML file named "12638-172-228_SeqListing.XML", created on July 20, 2023, and is 56,314 bytes in size. Technical Field

[0005] Provided herein are pharmaceutical compositions comprising antibodies that immunospecifically bind to the receptor tyrosine kinase KIT, and uses thereof. Also provided are kits for such pharmaceutical compositions and methods for preparing such pharmaceutical compositions. Background Art

[0006] 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 by Yarden et al., EMBO J., 1987, 6: 3341-3351. KIT is also known as CD117 or stem cell factor receptor ("SCFR") because it is the receptor for stem cell factor ("SCF") ligand (also known as Steel Factor or Kit ligand). Binding of the SCF ligand to the first three extracellular Ig-like domains of KIT induces receptor dimerization and thereby activates intrinsic tyrosine kinase activity through phosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains (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.

[0007] The fourth (D4) and fifth (D5) extracellular Ig-like domains of KIT are thought to mediate receptor dimerization (see, e.g., International Patent Application Publication No. WO 2008 / 153926; Yuzawa et al., Cell, 2007, 130:323-334).

[0008] Expression of KIT has been detected in a variety of cell types, such as mast cells, stem cells, brain cells, melanocytes, 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, L K., 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).

[0009] Antibodies that immunospecifically bind to human KIT are known, for example, from International Patent Publication No. WO2014018625A1, which is incorporated herein by reference in its entirety.

[0010] There is a need to provide suitable anti-human KIT antibody preparations. Summary of the Invention

[0011] In one aspect, provided herein is a pharmaceutical composition comprising: (i) an antibody that immunospecifically binds to human KIT, or an antigen-binding fragment thereof; (ii) a buffer; (iii) a salt; and (iv) an excipient.

[0012] In specific embodiments, the pH of the pharmaceutical composition is about 4 to about 7. In specific embodiments, the pH of the pharmaceutical composition is about 5 to about 6. In specific embodiments, the pH of the pharmaceutical composition is about 5.5.

[0013] In a specific embodiment, the salt is an alkali metal salt. In a specific embodiment, the alkali metal salt is sodium chloride. In a specific embodiment, the concentration of sodium chloride is about 25 mM to about 100 mM. In one embodiment, the concentration of sodium chloride is about 50 mM.

[0014] In a specific embodiment, the buffer is an alkali metal acetate. In a specific embodiment, the alkali metal acetate is sodium acetate. In a specific embodiment, the concentration of sodium acetate is about 1 mM to about 50 mM. In one embodiment, the concentration of sodium acetate is about 25 mM.

[0015] In a specific embodiment, the excipient is a sugar, a sugar alcohol or an amino acid. In a specific embodiment, the excipient is mannitol, sorbitol, sucrose, trehalose, glycine, arginine, alanine, histidine or any combination thereof. In a specific embodiment, the excipient is mannitol, sorbitol, sucrose, trehalose, glycine, arginine, histidine or any combination thereof. In a specific embodiment, the excipient is mannitol, sucrose, arginine, histidine or any combination thereof. In a specific embodiment, the excipient is mannitol. In a specific embodiment, the concentration of mannitol is about 1% to about 10%. In one embodiment, the concentration of mannitol is about 3%.

[0016] In a specific embodiment, the concentration of the antibody or its antigen-binding fragment is about 50 mg / ml to about 500 mg / ml. In a specific embodiment, the concentration of the antibody or its antigen-binding fragment is about 100 mg / ml to about 400 mg / ml. In one embodiment, the concentration of the antibody or its antigen-binding fragment is about 150 mg / ml.

[0017] In another aspect, provided herein is a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 50 mg / ml to about 500 mg / ml; (ii) an alkali metal acetate at a concentration of about 1 mM to about 50 mM; (iii) an alkali metal chloride at a concentration of about 25 mM to about 100 mM; and (iv) mannitol at a concentration of about 1% to about 10%; wherein the pH of the pharmaceutical composition is about 5 to about 6.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 50 mg / ml to about 500 mg / ml; (ii) an alkali metal acetate at a concentration of about 1 mM to about 50 mM; (iii) an alkali metal chloride at a concentration of about 25 mM to about 100 mM; and (iv) mannitol at a concentration of about 1% to about 10%; wherein the pH of the pharmaceutical composition is about 5 to about 6.

[0019] In another aspect, provided herein is a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 150 mg / ml; (ii) sodium acetate at a concentration of about 25 mM; (iii) sodium chloride at a concentration of about 50 mM; and (iv) mannitol at a concentration of about 3%; wherein the pH of the pharmaceutical composition is about 5.5.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0021] (A)(i) a light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0022] (ii) a heavy chain variable region ("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;

[0023] (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0024] (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively;

[0025] (C)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; and

[0026] (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 32, respectively;

[0027] (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0028] (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 27, respectively; or

[0029] (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and

[0030] (ii) VH, comprising VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.

[0031] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a VL comprising VL CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2-4, respectively; and a VH comprising VH CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 5-7, respectively.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a VL comprising the amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYSGVPDRFX K3GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X K1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 Is an amino acid with an aliphatic hydroxyl side chain or P, X K5 is an amino acid with a charged or acidic side chain, and X K6 is an amino acid having an aromatic side chain; and (ii) a VH comprising the following amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAPGKGLEWIARIYPGSGNTYYNEKFKGRX H4 TX HH5 TAX HH 6KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, 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 amino acid F or S, X K2 is amino acid A or S, X K3 is amino acid T or S, X K4 is amino acid S or P, X K5 is amino acid D or T, X K6 is amino acid F or Y, X H1 Is amino acid L or V, X H2 Is amino acid L or V, X H3 Is amino acid K or R, X H4 Is amino acid V or A, XH5 Is amino acid L or I, X H6 is amino acid E or D, X H7 is amino acid Q or E, and X H8 It is the amino acid S or T.

[0033] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, and 16; and a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12.

[0034] In a specific embodiment, the antibody comprises a human heavy chain constant region, and wherein the human heavy chain constant region is a human IgG1 constant region.

[0035] In certain embodiments, the antibody comprises a modified human Fc region or domain.

[0036] In a specific embodiment, the antibody comprises a modified human IgG1 Fc region or domain. In a specific embodiment, the modified human IgG1 Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q, and 322Q, numbered according to the EU index as set forth in Kabat. In another specific embodiment, the modified human IgG1 Fc region or domain further comprises the non-naturally occurring amino acids 252Y, 254T, and 256E, numbered according to the EU index as set forth in Kabat.

[0037] 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 human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q, and 322Q, numbered according to the EU index as set forth in Kabat.

[0038] 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 human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, numbered according to the EU index as set forth in Kabat.

[0039] In a specific embodiment, the antibody comprises a heavy chain comprising the following amino acid sequence:

[0040]

[0041] In a specific embodiment, the antibody comprises a light chain comprising the following amino acid sequence:

[0042]

[0043] In another aspect, provided herein is a kit comprising a pharmaceutical composition described herein.

[0044] In another aspect, provided herein is a method for preventing, treating, or managing a KIT-related disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition described herein.

[0045] In certain embodiments, the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In one embodiment, the KIT-associated disorder is a mast cell-associated disorder. In another embodiment, the KIT-associated disorder is an eosinophil-associated disorder.

[0046] In certain embodiments, the methods of treatment provided herein further comprise 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 immunomodulator, or an anti-inflammatory agent.

[0047] In certain embodiments, the subject is a human.

[0048] In a specific embodiment, the subject is administered an antibody or antigen-binding fragment thereof at a dose of ≥1.5 mg / kg per dose. In a specific embodiment, the subject is administered an antibody or antigen-binding fragment thereof at a dose of about 1.5 mg / kg to about 4.5 mg / kg per dose. In a specific embodiment, the subject is administered an antibody or antigen-binding fragment thereof at a dose of about 1.5 mg / kg per dose. In a specific embodiment, the subject is administered an antibody or antigen-binding fragment thereof at a dose of about 3.0 mg / kg per dose. In a specific embodiment, the subject is administered an antibody or antigen-binding fragment thereof at a dose of about 4.5 mg / kg per dose.

[0049] In a specific embodiment, two doses of the antibody or antigen-binding fragment thereof are administered to the subject. In a specific embodiment, three doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0050] In a specific embodiment, the antibody or antigen-binding fragment thereof is administered to the subject once every 4 weeks. In a specific embodiment, the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks.

[0051] In one embodiment, the subject is administered about 1.5 mg / kg of the antibody or antigen-binding fragment thereof per dose once every four weeks for three doses. In another embodiment, the subject is administered about 3.0 mg / kg of the antibody or antigen-binding fragment thereof per dose once every eight weeks for two doses. In another embodiment, the subject is administered about 4.5 mg / kg of the antibody or antigen-binding fragment thereof per dose once every eight weeks for two doses.

[0052] In another aspect, provided herein is a method for preparing a pharmaceutical composition described herein, the method comprising combining an antibody or antigen-binding fragment thereof with a buffer, salts, and an excipient.

[0053] 3.1 Exemplary Implementation

[0054] 1. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT; (ii) a buffer; (iii) a salt; and (iv) an excipient.

[0055] 2. The pharmaceutical composition of embodiment 1, wherein the pharmaceutical composition has a pH of about 4 to about 7.

[0056] 3. The pharmaceutical composition of embodiment 2, wherein the pharmaceutical composition has a pH of about 5 to about 6.

[0057] 4. The pharmaceutical composition of embodiment 3, wherein the pharmaceutical composition has a pH of about 5.5.

[0058] 5. The pharmaceutical composition of any one of the preceding embodiments, wherein the salt is an alkali metal salt.

[0059] 6. The pharmaceutical composition according to embodiment 5, wherein the alkali metal salt is sodium chloride.

[0060] 7. The pharmaceutical composition of embodiment 6, wherein the concentration of sodium chloride is about 25 mM to about 100 mM.

[0061] 8. The pharmaceutical composition of embodiment 7, wherein the concentration of sodium chloride is about 50 mM.

[0062] 9. The pharmaceutical composition of any one of the preceding embodiments, wherein the buffer is an alkali metal acetate.

[0063] 10. The pharmaceutical composition of embodiment 9, wherein the alkali metal acetate is sodium acetate.

[0064] 11. The pharmaceutical composition of embodiment 10, wherein the concentration of sodium acetate is about 1 mM to about 50 mM.

[0065] 12. The pharmaceutical composition of embodiment 11, wherein the concentration of sodium acetate is about 25 mM.

[0066] 13. The pharmaceutical composition of any one of the preceding embodiments, wherein the excipient is a sugar, a sugar alcohol, an amino acid, or any combination thereof.

[0067] 14. The pharmaceutical composition of embodiment 13, wherein the excipient is mannitol, sorbitol, sucrose, trehalose, glycine, arginine, alanine, histidine, or any combination thereof.

[0068] 15. The pharmaceutical composition of embodiment 13, wherein the excipient is mannitol, sorbitol, sucrose, trehalose, glycine, arginine, histidine, or any combination thereof.

[0069] 16. The pharmaceutical composition of embodiment 14 or 15, wherein the excipient is mannitol, sucrose, arginine, histidine, or any combination thereof.

[0070] 17. The pharmaceutical composition of any one of embodiments 13-16, wherein the excipient is mannitol.

[0071] 18. The pharmaceutical composition of embodiment 17, wherein the concentration of mannitol is about 1% to about 10%.

[0072] 19. The pharmaceutical composition of embodiment 18, wherein the concentration of mannitol is about 3%.

[0073] 20. The pharmaceutical composition of any one of the preceding embodiments, wherein the concentration of the antibody or antigen-binding fragment thereof is about 50 mg / ml to about 500 mg / ml.

[0074] 21. The pharmaceutical composition of embodiment 20, wherein the concentration of the antibody or antigen-binding fragment thereof is about 100 mg / ml to about 400 mg / ml.

[0075] 22. The pharmaceutical composition of embodiment 21, wherein the concentration of the antibody or antigen-binding fragment thereof is about 150 mg / ml.

[0076] 23. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 50 mg / ml to about 500 mg / ml; (ii) an alkali metal acetate at a concentration of about 1 mM to about 50 mM; (iii) an alkali metal chloride at a concentration of about 25 mM to about 100 mM; and (iv) mannitol at a concentration of about 1% to about 10%; wherein the pH of the pharmaceutical composition is about 5 to about 6.

[0077] 24. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 50 mg / ml to about 500 mg / ml; (ii) sodium acetate at a concentration of about 1 mM to about 50 mM; (iii) sodium chloride at a concentration of about 25 mM to about 100 mM; and (iv) mannitol at a concentration of about 1% to about 10%; wherein the pH of the pharmaceutical composition is about 5 to about 6.

[0078] 25. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 150 mg / ml; (ii) sodium acetate at a concentration of about 25 mM; (iii) sodium chloride at a concentration of about 50 mM; and (iv) mannitol at a concentration of about 3%; wherein the pH of the pharmaceutical composition is about 5.5.

[0079] 26. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises:

[0080] (A)(i) a light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0081] (ii) a heavy chain variable region ("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;

[0082] (B)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0083] (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively;

[0084] (C)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; and

[0085] (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 32, respectively;

[0086] (D)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0087] (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 27, respectively; or

[0088] (E)(i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and

[0089] (ii) VH, comprising VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.

[0090] 27. The pharmaceutical composition of any of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising VL CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 2-4, respectively; and a VH comprising VH CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 5-7, respectively.

[0091] 28. A pharmaceutical composition as described in any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises

[0092] (i) a VL comprising the following amino acid sequence:

[0093] DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSAS YRYSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKV EIK (SEQ ID NO: 17), wherein X K1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 Is an amino acid with an aliphatic hydroxyl side chain or P, X K5 is an amino acid with a charged or acidic side chain, and X K6 is an amino acid with an aromatic side chain; and

[0094] (ii) VH comprising the following amino acid sequence:

[0095] QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAPGKGLEWIARIYPGSGNTYYNEKFKGRXH4 TX H5 TAX H6 KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 It is an amino acid with an aliphatic hydroxyl side chain.

[0096] 29. The pharmaceutical composition according to embodiment 28, wherein X Kl is amino acid F or S, X K2 is amino acid A or S, X K3 is amino acid T or S, X K4 is amino acid S or P, X K5 is amino acid D or T, X K6 is amino acid F or Y, X H1 Is amino acid L or V, X H2 Is amino acid L or V, X H3 Is amino acid K or R, X H4 Is amino acid V or A, X H5 Is amino acid L or I, X H6 is amino acid E or D, X H7 is amino acid Q or E, and X H8 It is the amino acid S or T.

[0097] 30. The pharmaceutical composition of any of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, and 16; and a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12.

[0098] 31. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises a human heavy chain constant region, and wherein the human heavy chain constant region is a human IgG1 constant region.

[0099] 32. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises a modified human Fc region or domain.

[0100] 33. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises a modified human IgG1 Fc region or domain.

[0101] 34. A pharmaceutical composition as described in embodiment 33, wherein the modified human IgG1 Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q and 322Q numbered according to the EU index as shown in Kabat.

[0102] 35. A pharmaceutical composition as described in embodiment 34, wherein the modified human IgG1 Fc region or domain further comprises the non-naturally occurring amino acids 252Y, 254T and 256E numbered according to the EU index as shown in Kabat.

[0103] 36. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises:

[0104] (i) VL comprising the amino acid sequence of SEQ ID NO: 14;

[0105] (ii) VH comprising the amino acid sequence of SEQ ID NO: 10; and

[0106] (iii) a modified human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q and 322Q, numbered according to the EU index as set forth in Kabat.

[0107] 37. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises:

[0108] (i) VL comprising the amino acid sequence of SEQ ID NO: 14;

[0109] (ii) VH comprising the amino acid sequence of SEQ ID NO: 10; and

[0110] (iii) a modified human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E, numbered according to the EU index as set forth in Kabat.

[0111] 38. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises a heavy chain comprising the following amino acid sequence:

[0112]

[0113]

[0114] 39. The pharmaceutical composition of any one of the preceding embodiments, wherein the antibody comprises a light chain comprising the following amino acid sequence:

[0115]

[0116] 40. A pharmaceutical kit comprising the pharmaceutical composition of any one of the preceding embodiments.

[0117] 41. 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 pharmaceutical composition of any one of embodiments 1-39.

[0118] 42. The method of embodiment 41, wherein the pharmaceutical composition is administered subcutaneously to the subject.

[0119] 43. The method of embodiment 41 or 42, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.

[0120] 44. The method of embodiment 43, wherein the KIT-associated disorder is a mast cell-associated disorder.

[0121] 45. The method of embodiment 43, wherein the KIT-associated disorder is an eosinophil-associated disorder.

[0122] 46. ​​The method of any one of embodiments 41-45, further comprising administering a second therapeutic agent to the subject.

[0123] 47. The method of embodiment 46, 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.

[0124] 48. The method of any one of embodiments 41 to 47, wherein the subject is human.

[0125] 49. The method of any one of embodiments 41-48, wherein the subject is administered ≥ 1.5 mg / kg of the antibody or antigen-binding fragment thereof per dose.

[0126] 50. The method of any one of embodiments 41-48, wherein the subject is administered about 1.5 mg / kg to about 4.5 mg / kg of the antibody or antigen-binding fragment thereof per dose.

[0127] 51. The method of any one of embodiments 41-48, wherein the subject is administered about 1.5 mg / kg of the antibody or antigen-binding fragment thereof per dose.

[0128] 52. The method of any one of embodiments 41-48, wherein the subject is administered about 3.0 mg / kg of the antibody or antigen-binding fragment thereof per dose.

[0129] 53. The method of any one of embodiments 41-48, wherein the subject is administered about 4.5 mg / kg of the antibody or antigen-binding fragment thereof per dose.

[0130] 54. The method of any one of embodiments 41-53, wherein two doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0131] 55. The method of any one of embodiments 41-53, wherein three doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0132] 56. The method of any one of embodiments 41-55, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 4 weeks.

[0133] 57. The method of any one of embodiments 41-55, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks.

[0134] 58. The method of any one of embodiments 41-48, wherein the subject is administered about 1.5 mg / kg of the antibody or antigen-binding fragment thereof per dose once every four weeks for three doses.

[0135] 59. The method of any one of embodiments 41-48, wherein the subject is administered about 3.0 mg / kg of the antibody or antigen-binding fragment thereof per dose once every 8 weeks for a total of two doses.

[0136] 60. The method of any one of embodiments 41-48, wherein the subject is administered about 4.5 mg / kg of the antibody or antigen-binding fragment thereof per dose once every 8 weeks for a total of two doses.

[0137] 61. Use of the pharmaceutical composition of any one of embodiments 1-39 for the manufacture of a medicament for preventing, treating, or managing a KIT-associated disorder in a subject.

[0138] 62. The use according to embodiment 61, wherein the pharmaceutical composition is formulated for subcutaneous administration.

[0139] 63. The use of embodiment 61 or 62, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.

[0140] 64. The use according to embodiment 63, wherein the KIT-associated disorder is a mast cell-associated disorder.

[0141] 65. The use according to embodiment 63, wherein the KIT-associated disorder is an eosinophil-associated disorder.

[0142] 66. The use of any one of embodiments 61-65, wherein the medicament is manufactured to be administered to the subject in combination with a second therapeutic agent.

[0143] 67. The use of embodiment 66, 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.

[0144] 68. The use of any one of embodiments 61-67, wherein the subject is a human.

[0145] 69. The use of any one of embodiments 61-68, wherein ≥1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0146] 70. The use of any one of embodiments 61-68, wherein about 1.5 mg / kg to about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0147] 71. The use of any one of embodiments 61-68, wherein about 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0148] 72. The use of any one of embodiments 61-68, wherein about 3.0 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0149] 73. The use of any one of embodiments 61-68, wherein about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0150] 74. The use of any one of embodiments 61-73, wherein two doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0151] 75. The use of any one of embodiments 61-73, wherein three doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0152] 76. The use of any one of embodiments 61-75, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 4 weeks.

[0153] 77. The use of any one of embodiments 61-75, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks.

[0154] 78. The use of any one of embodiments 61-68, wherein each dose of about 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every four weeks for a total of three doses.

[0155] 79. The use of any one of embodiments 61-68, wherein each dose of about 3.0 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks for a total of two doses.

[0156] 80. The use of any one of embodiments 61-68, wherein each dose of about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks for a total of two doses.

[0157] 81. The pharmaceutical composition of any one of embodiments 1-39, for use in a method of preventing, treating, or managing a KIT-associated disorder in a subject.

[0158] 82. The pharmaceutical composition for use of embodiment 81, wherein the pharmaceutical composition is administered subcutaneously to the subject.

[0159] 83. The pharmaceutical composition for use of embodiment 81 or 82, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.

[0160] 84. The pharmaceutical composition for use of embodiment 83, wherein the KIT-associated disorder is a mast cell-associated disorder.

[0161] 85. The pharmaceutical composition for use of embodiment 83, wherein the KIT-associated disorder is an eosinophil-associated disorder.

[0162] 86. The pharmaceutical composition for use of any one of embodiments 81-85, wherein the method further comprises administering a second therapeutic agent to the subject.

[0163] 87. The pharmaceutical composition for use of embodiment 86, 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.

[0164] 88. The pharmaceutical composition for use of any one of embodiments 81-87, wherein the subject is a human.

[0165] 89. The pharmaceutical composition for use of any one of embodiments 81-88, wherein ≥ 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0166] 90. The pharmaceutical composition for use of any one of embodiments 81-88, wherein about 1.5 mg / kg to about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0167] 91. The pharmaceutical composition for use of any one of embodiments 81-88, wherein about 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0168] 92. The pharmaceutical composition for use of any one of embodiments 81-88, wherein about 3.0 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0169] 93. The pharmaceutical composition for use of any one of embodiments 81-88, wherein about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

[0170] 94. The pharmaceutical composition for use of any one of embodiments 81-93, wherein two doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0171] 95. The pharmaceutical composition for use of any one of embodiments 81-93, wherein three doses of the antibody or antigen-binding fragment thereof are administered to the subject.

[0172] 96. The pharmaceutical composition for use of any one of embodiments 81-95, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 4 weeks.

[0173] 97. The pharmaceutical composition for use of any one of embodiments 81-95, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks.

[0174] 98. The pharmaceutical composition for use of any one of embodiments 81-88, wherein each dose of about 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every four weeks for three doses.

[0175] 99. The pharmaceutical composition for use of any one of embodiments 81-88, wherein each dose of about 3.0 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks for a total of two doses.

[0176] 100. The pharmaceutical composition for use of any one of embodiments 81-88, wherein each dose of about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks.

[0177] 101. A method for preparing the pharmaceutical composition of any one of embodiments 1-39, the method comprising combining the antibody or antigen-binding fragment thereof with a buffer, salt, and an excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figure 1 The amino acid sequence of full-length human KIT is depicted (SEQ ID NO: 1), GenBank TM The accession number is AAC50969. The first to fifth extracellular Ig-like domains (i.e., D1, D2, D3, D4, and D5) are indicated; "{" delineates the amino-terminal residue of each domain, and "}" delineates the carboxyl-terminal residue of each domain. The D1 domain is depicted at P34 to R112, the D2 domain is depicted at D113 to P206, the D3 domain is depicted at A207 to D309, the D4 domain is depicted at K310 to N410, the hinge region between D4 and D5 is located at V409 to N410, and the D5 domain is depicted at T411 to K509. In addition, 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 comprises K310 to K509, the transmembrane domain comprises residues F525 to Q545, and the kinase domain comprises residues K589 to S933.

[0179] Figures 2A-2E The effect of a specific anti-KIT antibody mAb1 according to the invention on plasma tryptase levels is depicted.

[0180] Figure 3A and3B The effect of a specific anti-KIT antibody mAb1 according to the invention on plasma tryptase levels is depicted.

[0181] Figure 4 The effect of a specific anti-KIT antibody mAb1 according to the present invention on plasma stem cell factor (SCF) levels is depicted.

[0182] Figure 5 The effects of a specific anti-KIT antibody mAb1 according to the present invention and a corresponding antibody mAbc having the same variable region sequence but unmutated (wild-type) human IgG1 sequence on SCF-induced wild-type KIT activation and downstream intracellular signaling pathways are depicted.

[0183] Figure 6 Depicted are the effects of a specific anti-KIT antibody mAb1 according to the invention and the corresponding antibody mAbc having the same variable region sequence but unmutated (wild-type) human IgG1 sequence on SCF-dependent cell proliferation.

[0184] Figure 7 The binding affinities of a specific anti-KIT antibody mAb1 according to the present invention and the corresponding antibody mAbc having the same variable region sequence but not mutated (wild type) human IgG1 sequence to recombinant human Fc-γ receptor (FcγR) and human neonatal Fc receptor (FcRn) are shown.

[0185] Figures 8A-8N Binding curves of the specific anti-KIT antibody mAb1 according to the present invention and the corresponding antibody mAbc having the same variable region sequence but unmutated (wild-type) human IgG1 sequence to recombinant human Fc-γ receptor (FcγR) and human neonatal Fc receptor (FcRn) are depicted. Figure 8A The binding curve of mAb1 to FcγRI is depicted. Figure 8B The binding curve of mAb1 to FcγRIIa is depicted. Figure 8C Binding curves of mAbl to FcγRIIb are depicted. Figure 8D Binding curves of mAbl to FcγRIIIa are depicted. Figure 8E The binding curve of mAb1 to FcγRIIIb is depicted. Figure 8F The binding curve of mAb1 to FcRn (pH 6.0) is depicted. Figure 8G The binding curve of mAb1 to FcRn (pH 7.2) is depicted. Figure 8H Binding curves of mAbc to FcγRI are depicted. Figure 8I Binding curves of mAbc to FcγRIIa are depicted. Figure 8J Binding curves of mAbc to FcγRIIb are depicted. Figure 8KBinding curves of mAbc to FcγRIIIa are depicted. Figure 8L Binding curves of mAbc to FcγRIIIb are depicted. Figure 8M The binding curve of mAbc to FcRn (pH 6.0) is depicted. Figure 8N Binding curves of mAbc to FcRn (pH 7.2) are depicted.

[0186] Figure 9 Depicted are the effects of a specific anti-KIT antibody mAb1 according to the invention and the corresponding antibody mAbc having the same variable region sequence but unmutated (wild-type) human IgG1 sequence on antibody-dependent cellular cytotoxicity (ADCC) activity.

[0187] Figure 10 Depicted are the effects of specific anti-KIT antibodies mAb1 according to the present invention on specific cytokine production. Each bar graph shows the following conditions from left to right: PHA, LPS, huIgG1 (soluble), 0.02 nM mAb1 (soluble), 0.2 nM mAb1 (soluble), 40 nM mAb1 (soluble), 0.02 nM mAb1 (dry coated), 0.2 nM mAb1 (dry coated), and 40 nM mAb1 (dry coated).

[0188] Figure 11 Depicted is a schematic diagram showing the role of KIT signaling in mast cells and the effect of mAbl on the KIT receptor.

[0189] Figures 12A-12D A single dose of mAb1 induced rapid and durable responses in patients with chronic induced urticaria (CIndU), with a complete remission (CR) rate of 95%. Ten of ten patients with cold urticaria (ColdU) achieved CR ( Figure 12A ). 8 / 9 symptomatic skin scratching (SD) patients achieved CR, and 1 / 9 SD patients achieved partial remission (PR) ( Figure 12B CR = negative challenge test, ≤4°C or 0 injections; PR = improvement, 4°C or ≥2 injections; the maximum response for each patient is shown. Figure 12C Figure 2 shows the changes in ColdU patients over time. Results: Among ColdU patients who completed treatment (n=8), the median duration of CR was 77 days ( Figure 12C ). Figure 12D Figure 2 shows the changes in SD patients over time. Results. Among SD patients who completed treatment (n=6), the median duration of CR was 57 days ( Figure 12D ).

[0190] Figures 13A-13B Overall disease improvement as confirmed by Physician's Global Assessment (Phys-GA) and Patient's Global Assessment (Pat-GA) is shown. Phys-GA and Pat-GA assess disease severity using a 0-3 Likert scale, where 0 represents no disease and 3 represents severe disease.

[0191] Figures 14A-14D mAb1 treatment significantly depleted skin mast cells and serum tryptase. Figure 14A It was shown that mAbl reduced the number of skin mast cells (n=14, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001). Figure 14B It was shown that mAbl reduced serum tryptase to below detection levels in all patients (tryptase values ​​below the limit of quantitation (LLoQ = 1 ng / mL) of the assay were normalized to 0). Figure 14C Mast cell and tryptase kinetics are shown. Figure 14D The results showed that the number of skin mast cells was correlated with the serum tryptase level (p < 0.0001; R 2 =0.45)).

[0192] Figures 15A-15D showed that the kinetics of skin mast cell and tryptase depletion reflected a lowered threshold for excitation. Figure 15A shows mast cell dynamics and Changes in results over time. Figure 15B Shows the dynamics of mast cells in SD patients and Changes in results over time. Figure 15C Shown are the tryptase kinetics and Results over time (tryptase values ​​below the LLoQ were normalized to 0; critical temperature thresholds below 4°C (negative test) were assigned a value of 3°C). Figure 15D Shows the dynamics of mast cells in SD patients and Changes in results over time.

[0193] Figures 16A-16D Hematological parameters generally remained within normal ranges, with mild, transient, and asymptomatic decreases in hemoglobin and white blood cell (WBC) parameters observed. Figure 16A Shows changes in hemoglobin (HgB) levels over time. Figure 16B Displays the WBC count over time. Figure 16C Shows changes in platelet count over time. Figure 16DFigure 2. Changes in absolute neutrophil count (ANC) over time. In each graph, the shaded area represents the corresponding normal range.

[0194] Figures 17A-17B A single 3 mg / kg dose of mAb1 was shown to be effective in patients with cold urticaria (ColdU) (n=10, see Figure 17A ) and symptomatic dermatographism (SD) patients (n = 10, see Figure 17B ) caused rapid and sustained improvement in urticaria control. An Urticaria Control Test (UCT) score of 16 indicates complete control of urticaria, a UCT score of ≥12 indicates good control of urticaria, and a UCT score of <12 indicates poor control of urticaria. In each figure, the mean UCT score ± SEM is shown.

[0195] Figures 18A-18B showed that a single 3 mg / kg dose of mAbl caused rapid and sustained improvement in urticaria control in both ColdU and SD patients. Figure 18A The results showed that by week 8, 100% of patients achieved "good control" status (UCT score ≥ 12). Figure 18B It showed that by week 8, 63% of patients achieved "complete control" status (UCT score = 16).

[0196] Figures 19A-19B mAb1 significantly reduced the incidence of cold urticaria (ColdU, n = 10, see Figure 19A ) and symptomatic dermatographism (SD, n = 10, see Figure 19B ) Impact on patients' quality of life. Mean DLQI scores ± SEM are shown.

[0197] Figures 20A-20B mAb1 was shown to significantly reduce the impact of the disease on the quality of life of patients with cold urticaria (ColdU) and symptomatic dermatographism (SD). Figure 20A Results showed that by week 4, 93% of patients achieved clinically significant improvement in quality of life. : A DLQI reduction of ≥4 points is the minimal clinically important difference (MCID). *: Only patients with a baseline DLQI score of ≥4 are included. Figure 20B showed that by week 4, 58% of patients reported no impact of the disease on their quality of life. : Includes all responses provided each week.

[0198] Figures 21A-21B It was shown that a single 3 mg / kg dose of mAbl induced rapid and durable improvement in the challenge test, with a complete remission (CR) rate of 95% and a significant reduction in tryptase. Figure 21AmAb1 was shown to induce rapid and durable improvements in provocative testing, with a complete remission rate of 95%. The critical temperature threshold (CTT) of cold urticaria (ColdU) was determined and the Disease activity was assessed using the critical friction threshold (CFT) for symptomatic dermatographism (SD). *: A critical temperature threshold below 4°C (negative test) was assigned a 3°C value. In the study, 10 / 10 ColdU patients and 9 / 10 SD patients experienced CR. CR = negative provocative test, ≤4°C (for ColdU) or 0 needles (for SD). In the figures, mean ± SEM are shown. Figure 21B It was shown that mAb 1 caused a rapid, sustained and significant reduction in tryptase. Tryptase values ​​below the limit of quantification (1 ng / mL) were normalized to 0. In the figure, mean ± SEM are shown.

[0199] Figure 22 Describes the study design of a Phase 1 study in adults with moderate to severe chronic spontaneous urticaria (CSU).

[0200] Figures 23A-23C It was shown that mAbl induces rapid and sustained improvement of symptoms in patients with CSU refractory to antihistamines.The data presented are means ± SE. Figure 23A Data for UAS7 (Weekly Urticaria Activity Score) are shown. Figure 23B Data for ISS7 (Weekly Itch Severity Score) are shown. Figure 23C Data for HSS (Weekly Urticaria Severity Score) are shown.

[0201] Figures 24A-24B showed that mAb1 elicited durable responses at doses ≥1.5 mg / kg, based on UAS7. Figure 24A Data are shown for the percentage of patients with UAS7≤6. Figure 24B Data are shown for the percentage of patients with UAS7=0.

[0202] Figures 25A-25B It was shown that prolonged mAbl exposure and tryptase inhibition were achieved at doses ≥ 1.5 mg / kg. Figure 25A Pharmacokinetic data are shown (data presented are geometric means ± geometric standard deviations). Figure 25B Serum tryptase data are shown (data presented are mean ± SE; tryptase values ​​below the limit of detection were normalized to 0).

[0203] Figures 26A-26B It was shown that better urticaria disease control (UCT≥12) was achieved using mAbl at doses ≥1.5 mg / kg. Figure 26A Mean UCT scores are shown (data presented are mean ± SE). Figure 26B Shows the percentage of patients with UCT ≥ 12. UCT = 16: Complete control. UCT ≥ 12: Well controlled.

[0204] Figures 27A-27B Strong clinical activity was observed in both omalizumab-treated and untreated patients. Figure 27A Showing average UAS7. Figure 27B Mean UCT is shown. Data presented are mean ± SE.

[0205] Figures 28A-28D Changes in key hematological parameters over time are shown. Figure 28A : Hemoglobin. Figure 28B :leukocyte. Figure 28C : Neutrophils. Figure 28D : Platelets. Data presented are mean ± SE. DETAILED DESCRIPTION

[0206] Anti-KIT antibody formulations are provided herein. Specifically, pharmaceutical compositions are provided herein, comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT; (ii) a buffer; (iii) a salt; and (iv) an excipient. Methods for preparing such pharmaceutical compositions are also provided. Also provided herein are methods and uses for preventing, treating, or managing KIT-related conditions or diseases, comprising administering the pharmaceutical compositions described herein. Also provided herein are kits comprising the pharmaceutical compositions described herein.

[0207] As used herein, "administer" 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, or a pharmaceutical composition described herein) to a subject or patient (e.g., a human), such as by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0208] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a therapeutic amount (e.g., an antibody or pharmaceutical composition provided herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease and / or its associated symptoms. These terms also encompass the amount required to reduce, slow, or ameliorate the development or progression of a given disease, reduce, slow, or ameliorate the recurrence, development, or onset of a given disease, and / or improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than an anti-KIT antibody or pharmaceutical composition provided herein). In some embodiments, an "effective amount" as used herein also refers to an amount of an antibody or pharmaceutical composition described herein that achieves a specified result, such as a decrease in the number and / or activity of mast cells, a decrease in the number and / or activity of eosinophils, inhibition (e.g., partial inhibition) of a cellular KIT biological activity, such as inhibition of cell proliferation or cell survival, or enhancement or induction of apoptosis or cell differentiation, etc.

[0209] As used herein, the term "D4 or D5 region" or "D4 / D5 domain" refers to the region of 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 ("D4-D5 hinge region") of KIT, in the order from amino terminus to carboxyl terminus: D4, D4-D5 hinge region, and D5. As used herein, Figure 1 Amino acids V308 to H515 in are considered an example of a D4 / D5 region or domain.

[0210] As used herein, the term "KIT" or "KIT receptor" or "KIT polypeptide" refers to any form of full-length KIT, including but not limited to native KIT, KIT isoforms, interspecies KIT homologs, or KIT variants, such as naturally occurring (e.g., allelic or splice variants, or mutants, such as somatic mutants) or artificially constructed variants (e.g., recombinant or chemically modified variants). 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 TM Accession No. NM 000222 provides an exemplary human KIT nucleic acid sequence. TMAccession numbers NP 001087241, PI0721, and AAC50969 provide exemplary human KIT amino acid sequences. TM An exemplary mouse KIT amino acid sequence is provided in Accession No. AAH75716. Native KIT comprises 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). Figure 1 An exemplary amino acid sequence of the D4 / D5 region of human KIT, i.e., amino acid residues V308 to H515, is provided in . In a specific embodiment, KIT is human KIT. In a particular embodiment, KIT can exist as a monomer, dimer, multimer, native form, or denatured form.

[0211] As used herein, the term "combination" refers to the use of more than one therapy in the context of administering other therapies. The use of the term "combination" does not limit the order in which the therapies are administered. The therapies can be administered, for example, serially, sequentially, simultaneously, or concurrently.

[0212] As used herein, the terms "KIT-related disorder" or "KIT-related disease" are used interchangeably and mean any disease that is caused, associated with, or results in whole or in part from KIT expression and / or its activity or lack thereof. In a preferred embodiment, a KIT-related disorder or disease is a disease that is caused, associated with, or results in whole or in part from KIT expression and / or activity (e.g., KIT overexpression, gain-of-function KIT activity, and / or increased KIT activity). In a specific embodiment, a KIT-related disease or condition is a disease that is associated with KIT expression and / or activity, e.g., a disease that involves cells expressing KIT and / or exhibiting KIT activity, but is not caused or contributed to by KIT expression or activity. In one aspect, a KIT-related disorder or disease may be known to or may be determined by one skilled in the art. In a certain embodiment, a KIT-related disease or condition 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., a mutation or expression and / or activity of another gene) to contribute to the development and / or progression of a KIT-related disease or condition. In certain embodiments, the KIT-associated disease or disorder is associated with one or more mutations in KIT.

[0213] In certain embodiments, the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In certain embodiments, the KIT-associated disorder is a fibrotic or inflammatory disease, such as an inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC). In other embodiments, the KIT-associated disease is a cancer, such as lung cancer (e.g., small cell lung cancer), leukemia, neuroblastoma, melanoma, sarcoma (e.g., Ewing's sarcoma), or gastrointestinal stromal tumor (GIST). 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).

[0214] As used herein, the term "chronic prurigo" refers to a disease characterized by chronic pruritus (itching) and the presence of multiple localized or systemic prurigo lesions.

[0215] As used herein, the term "prurigo nodularis" refers to a disease characterized by chronic pruritus and the presence of multiple localized or generalized, raised, firm, and nodular lesions.

[0216] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or improvement in the progression, severity, and / or duration of a KIT-associated 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, such as the antibodies or pharmaceutical compositions provided herein.

[0217] As used herein, the terms "manage," "managing," and "management" refer to the beneficial effects a subject obtains from a therapy (e.g., a prophylactic or therapeutic agent) that does not result in a cure. In certain embodiments, one or more therapies (e.g., a prophylactic or therapeutic agent, such as an antibody or pharmaceutical composition described herein) are administered to a subject to "manage" a disorder or one or more symptoms thereof, thereby preventing the progression or worsening of the disorder.

[0218] As used herein, the terms "prevent," "impede," or "impeding," in the context of a disorder, refer to a complete or partial inhibition (e.g., less than 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or blocking of the development, recurrence, onset, or spread of a disorder and / or its associated symptoms resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody or pharmaceutical composition described herein).

[0219] As used herein, the term "prophylactic agent" refers to any agent that is capable of completely or partially inhibiting the development, recurrence, onset, or spread of a disorder and / or its associated symptoms in a subject. In certain embodiments, the term "prophylactic agent" refers to an antibody or pharmaceutical composition described herein. In certain other embodiments, the term "prophylactic agent" refers to an agent other than an antibody or pharmaceutical composition described herein. Generally, a prophylactic agent is an agent that is known to be useful, has been used, or is currently being used to prevent a disorder and / or its associated symptoms, or to arrest the onset, development, progression, and / or severity of a disorder and / or its associated symptoms. In specific embodiments, the prophylactic agent is a human anti-KIT antibody, such as a humanized or fully human anti-KIT monoclonal antibody, or a pharmaceutical composition thereof.

[0220] As used herein, the term "side effect" or "adverse effect" encompasses unwanted and adverse effects of a therapy (e.g., a preventive or therapeutic agent). An unwanted effect is not necessarily an adverse effect. An adverse effect of a therapy (e.g., a preventive or therapeutic agent) may be harmful or uncomfortable or risky. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth and loss of appetite, rash or swelling at the application site, flu-like symptoms such as fever, chills and fatigue, digestive tract problems, and allergic reactions. Other undesirable reactions experienced by patients are numerous and known in the art. Many such reactions are described in Physician's Desk Reference (71st edition, 2017).

[0221] As used herein, the terms "subject" and "patient" are used interchangeably herein. As used herein, a subject is a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, goat, rabbit, rat, mouse, etc.) or a primate (e.g., a monkey and a human), 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-related 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 child. In a specific embodiment, the subject is a child between the ages of 1 and 18 years old. In a specific embodiment, the subject is a human between the ages of 1 and 3 years old. In a specific embodiment, the subject is a human between the ages of 3 and 12 years old or 12 to 18 years old.

[0222] As used herein, the terms "therapies" and "therapy" may refer to any regimen, method, composition, formulation, and / or agent that can be used to prevent, prophylactically, treat, manage, or ameliorate a disease or condition, 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, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and / or other therapy used to prevent, treat, manage, prevent, or ameliorate a disease or condition, 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 described herein or a pharmaceutical composition described herein. In specific embodiments, "additional therapy" and "additional therapies" refer to a therapy other than treatment with an anti-KIT antibody described herein or a pharmaceutical composition described herein. In a specific embodiment, therapy comprises the use of an anti-KIT antibody or pharmaceutical composition described herein as an adjuvant therapy. For example, the anti-KIT antibodies or pharmaceutical compositions described herein are used in combination with drug therapy, biological therapy, surgery, and / or supportive care.

[0223] As used herein, the term "therapeutic agent" refers to any agent that can be used to prevent, treat, manage, or ameliorate a disorder and / or its associated symptoms. In certain embodiments, the term "therapeutic agent" refers to an anti-KIT antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody or pharmaceutical composition described herein. In specific embodiments, a therapeutic agent is an agent that is known to be useful, has been used, or is currently being used to prevent, treat, manage, or ameliorate a disorder or one or more symptoms associated therewith.

[0224] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein.

[0225] It should be understood that whenever aspects are described herein with the wording "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0226] As used herein, and unless otherwise indicated, the terms "about" and "approximately" should be interpreted to allow for normal variations within the judgment of one skilled in the art, such as variations within 20% or 10% or 5%. In specific embodiments, the terms "about" and "approximately" encompass the exact value recited.

[0227] 5.1 Antibodies

[0228] Provided herein are extracellular domains that specifically bind to a KIT receptor (e.g., a human KIT receptor, e.g., SEQ ID NO: 1 or Figure 1 ), or an antigen-binding fragment thereof.

[0229] As used herein, the terms "antibody," "immunoglobulin," and "Ig" are terms of art and are used interchangeably herein to refer to molecules that have an antigen binding site that immunospecifically binds to an antigen. The term "antibody" includes antigen-binding fragments.

[0230] Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain-antibody heavy chain pairs, heterologous conjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragments (scFv), camelized antibodies, affibodies (affybodies), Fab fragments, F(ab') fragments, disulfide bond-linked variable fragments (dsFv) and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody groups. Antibodies can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a or IgG2b). In certain embodiments, the antibodies described herein are IgG antibodies, or a class (eg, human IgG1 or IgG4) or subclass thereof.

[0231] As used herein, an "antigen" is a portion or molecule that contains an epitope and is therefore also specifically bound by an antibody. In a specific embodiment, the antigen bound by an antibody described herein 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).

[0232] As used herein, the terms "antigen binding domain," "antigen binding region," "antigen binding fragment," and similar terms refer to a portion of an antibody molecule that comprises amino acid residues that interact with an antigen and confer specificity to the antibody molecule for the antigen (e.g., complementary determining region (CDR)). The antigen binding region can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans. The CDRs of an antibody molecule can be determined using any method well known to those skilled in the art. Specifically, CDRs can 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 aspects, the CDRs of an antibody can be identified according to: (i) the Chothia numbering scheme, which will be 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, e.g., as described 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 AbM numbering system, e.g., as described in MacCallum et al., 1996, J. Mol. Biol, 262:732-745 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001); or (iv) the Contact numbering system, which is based on analysis of available complex crystal structures (bioinf.org.uk / abs) (see, e.g., MacCallum et al., (1996) J Mol Biol 5:732-745).In preferred embodiments, the antigen-binding fragments described herein comprise a full-length heavy chain Fc region or domain (e.g., a full-length human IgG1, human IgG2, human IgG3, or human IgG4 Fc region or domain) or a partial heavy chain Fc region or domain (e.g., a partial human IgG1, human IgG2, human IgG3, or human IgG4 Fc region or domain).

[0233] As used herein, the term "constant region" or "constant domain" refers to the portion of an antibody, such as the carboxyl terminal portion of a light chain and / or a heavy chain, that is not directly involved in binding the antibody to an antigen but exhibits or contributes to various effector functions, such as interaction with Fc receptors. The term refers to a portion of an immunoglobulin molecule that has an amino acid sequence that is generally more conserved than the immunoglobulin variable domain.

[0234] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which an antibody can specifically bind. The region or polypeptide constituting the epitope can be a continuous amino acid of a polypeptide, or the epitope can be composed of two or more discontinuous regions of a polypeptide.

[0235] As used herein, the term "heavy chain" when applied to antibodies refers to any of the different types, such as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), based on the amino acid sequence of the constant domain, thereby giving rise to IgA, IgD, IgE, IgG, and IgM class antibodies, respectively, including IgG subclasses such as IgGi, IgG2, IgG3, and IgG4. In a specific embodiment, the heavy chain is a human heavy chain.

[0236] As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex), and such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, as determined by, for example, immunoassays, Biacore, or other methods. TM The binding affinity of a molecule that immunospecifically binds to an antigen is generally lower than the affinity of the molecule that immunospecifically binds to an antigen. a In another embodiment, the molecule that immunospecifically binds to the antigen does not cross-react with other proteins. In another embodiment, the molecule that immunospecifically binds to the antigen does not cross-react with other non-KIT proteins.

[0237] As used herein, an "isolated" or "purified" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the antibody was derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, the antibodies or antigen-binding fragments described herein are isolated.

[0238] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in 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, Fifth Edition, 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 to 35 ("CDR1"), amino acid positions 50 to 65 ("CDR2"), and amino acid positions 95 to 102 ("CDR3"). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

[0239] As used herein, the term "light chain" when used with respect to antibodies refers to any of the different types, such as K (kappa) or λ (lambda), 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.

[0240] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a homogeneous or substantially homogeneous population of antibodies, and each monoclonal antibody typically recognizes a single epitope on an antigen. The term "monoclonal" is not limited to any particular method for antibody production. Generally, a monoclonal antibody population can be produced by a cell, a cell population, or a cell line. In specific embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell (e.g., a recombinant antibody-producing host cell), wherein the antibody immunospecifically binds to a KIT epitope (e.g., the D4 epitope of human KIT) as determined, for example, by ELISA or other antigen binding or competitive binding assays known in the art or as described in the Examples herein. The monoclonal antibodies described herein can be prepared, for example, by the hybridoma method described in Kohler et al.; Nature, 256:495 (1975), or can be isolated from phage libraries using, for example, the techniques described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed therefrom are well known in the art (see, for example, Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel et al., eds., John Wiley and Sons, New York). In a specific embodiment, the monoclonal antibodies are monospecific antibodies in that their antigen-binding regions are specific for the same epitope. In other specific embodiments, the monoclonal monospecific antibodies can be monovalent (having one antigen-binding region) or multivalent (having more than one antigen-binding region), e.g., bivalent (having two antigen-binding regions).

[0241] As used herein, the term "naked antibody" refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), a peptide or a polypeptide. In a specific embodiment, the naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, when the naked antibody is expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylase), the naked antibody will not be glycosylated. In certain embodiments, the naked antibody is a complete antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of a complete antibody, such as a Fab antibody.

[0242] As used herein, the term "polyclonal antibody" refers to an antibody population comprising a plurality of different antibodies directed against the same and different epi-positions in one or more antigens. Methods for producing polyclonal antibodies are known in the art (see, for example, Short Protocols in Molecular Biology, (2002) 5th edition, compiled by Ausubel et al., John Wiley and Sons, New York, Chapter 11).

[0243] As used herein, the term "recombinant human antibody" includes human antibodies that are isolated, prepared, expressed, or produced by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant, combinatorial human antibody library; antibodies isolated from animals that are transgenic and / or transchromosomal for human immunoglobulin genes (e.g., mice, rabbits, goats, or cows) (see, e.g., Taylor, L. D. et al., Nucl. Acids Res. 20: 6287-6295 (1992)); or antibodies prepared, expressed, produced, or isolated by any other means, such as by synthesis, genetic engineering of DNA sequences encoding human immunoglobulin sequences, or splicing of sequences encoding human immunoglobulins (e.g., human immunoglobulin gene sequences) into other such sequences. These recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, the amino acid sequences of these recombinant human antibodies are modified such that the amino acid sequences of the VH and / or VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may 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 a composite human sequence of the recombinant human antibody.

[0244] As used herein, the terms "variable region" and "variable domain" refer to a portion of an antibody, generally a portion of a light chain or a heavy chain, typically the amino-terminal approximately 110 to 120 amino acids in a mature heavy chain and the amino-terminal approximately 90 to 100 amino acids in a mature light chain, the sequence of which varies greatly from antibody to antibody and is used in the binding and specificity of a particular antibody to its specific antigen. Sequence variation is concentrated in a region called the complementarity determining region (CDR), while the more conserved region in the variable domain is called the framework region (FR).

[0245] 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 responsible for the interaction of the antibody with the antigen. In a specific embodiment, the numbering of amino acid positions in the antibodies described herein is according to the EU index, as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 ("Kabat et al."). In certain aspects, the CDRs of an antibody can be identified according to: (i) the Chothia numbering scheme, which will be 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, e.g., as described 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 AbM numbering system, e.g., as described in MacCallum et al., 1996, J. Mol. Biol, 262:732-745 and Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001); or (iv) the Contact numbering system, which is based on 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 murine CDRs and human framework regions (FRs). In specific 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 (FRs).As a non-limiting example, the variable regions described herein are obtained by assembling two or more human sequence fragments into a composite human sequence.

[0246] In a specific aspect, the anti-KIT antibodies (e.g., humanized antibodies) provided herein comprise a light chain variable region ("VL") comprising VL CDRs 1-3 and a heavy chain variable region ("VH") comprising VH CDRs 1-3, as shown in Table 1. In a specific aspect, the anti-KIT antibodies (e.g., humanized antibodies) provided herein comprise a light chain variable region ("VL") comprising VL CDRs 1-3 and a heavy chain variable region ("VH") comprising VH CDRs 1-3, as shown in Table 2 (Set 1 or Set 2). In a specific aspect, the anti-KIT antibodies (e.g., humanized antibodies) provided herein comprise a light chain variable region ("VL") comprising VL CDRs 1-3 and a heavy chain variable region ("VH") comprising VH CDRs 1-3, as shown in Table 3 (AbM CDRs or Contact CDRs).

[0247] In a specific aspect, the anti-KIT antibodies (e.g., humanized antibodies) provided herein comprise a VL comprising VL CDRs 1-3 (SEQ ID NOs: 2-4) as shown in Table 1 and a VH comprising VH CDRs 1-3 (SEQ ID NOs: 5-7) as shown in Table 1. In a specific embodiment, the anti-KIT antibody is a naked antibody. In a specific embodiment, the anti-KIT antibody is a bivalent monospecific antibody. In a specific embodiment, the anti-KIT antibody is a bispecific antibody. In a certain embodiment, the anti-KIT antibody is not a bispecific antibody.

[0248] Table 1: CDR amino acid sequences

[0249] Amino acid sequence SEQ ID NO: VL CDR1 KASQNVRTNVA 2 VL CDR2 SASYRYS 3 VL CDR3 QQYNSYPRT 4 VH CDR1 DYYIN 5 VH CDR2 RIYPGSGNTYYNEKFKG 6 VH CDR3 GVYYFDY 7

[0250] Table 2: CDR amino acid sequences

[0251]

[0252] Table 3: CDR amino acid sequences

[0253]

[0254] In a specific aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises:

[0255] (i) VL comprising the following amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2LIYSASYRYSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X K1 To X K6 is any amino acid; and

[0256] (ii) VH comprising the following amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAPGKGLEWIARIYPGSGNTYYNEKFKGRX H4 TX H5 TAX H6 KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X H1 To X H8 Any amino acid.

[0257] In a specific embodiment, X κ1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X κ2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X κ3 is an amino acid with an aliphatic hydroxyl side chain, X κ4 Is an amino acid with an aliphatic hydroxyl side chain or P, X K5 is an amino acid with a charged or acidic side chain, X K6 is an amino acid with an aromatic side chain, X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 It is an amino acid with an aliphatic hydroxyl side chain.

[0258] In a specific embodiment, X K1 is amino acid F or S, Xκ2 is amino acid A or S, X K3 is amino acid T or S, X K4 is amino acid S or P, X K5 is amino acid D or T, X K6 is amino acid F or Y, X H1 Is amino acid L or V, X H2 Is amino acid L or V, X H3 Is amino acid K or R, X H4 Is amino acid V or A, X H5 Is amino acid L or I, X H6 is amino acid E or D, X H7 is amino acid Q or E, and X H8 It is the amino acid S or T.

[0259] In a specific aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises:

[0260] (i) VL comprising the following amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X K1 To X K6 is any amino acid; and

[0261] (ii) VH, comprising VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0262] In a specific embodiment, X κ1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X κ2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X κ3 is an amino acid with an aliphatic hydroxyl side chain, X K4 Is an amino acid with an aliphatic hydroxyl side chain or P, X K5 is an amino acid with a charged or acidic side chain, and X K6 It is an amino acid with an aromatic side chain.

[0263] In a specific embodiment, X K1 is amino acid F or S, X κ2 is amino acid A or S, X K3 is amino acid T or S, X K4 is amino acid S or P, X K5 is amino acid D or T, and X K6 is amino acid F or Y.

[0264] In a specific aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises:

[0265] (i) a VL comprising VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2, and VL CDR3 have the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and

[0266] (ii) VH comprising the following amino acid sequence:

[0267] QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX HH3 QAPGKGLEWIARIYPGSGNTYYNEKFKGRX H4 TX H5 TAX H6 KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X H1 To X H8 is any amino acid.

[0268] In a specific embodiment, X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 It is an amino acid with an aliphatic hydroxyl side chain.

[0269] In a specific embodiment, X H1Is amino acid L or V, X H2 Is amino acid L or V, X H3 Is amino acid K or R, X H4 Is amino acid V or A, X H5 Is amino acid L or I, X H6 is amino acid E or D, X H7 is amino acid Q or E, and X H8 It is the amino acid S or T.

[0270] In a specific aspect, 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 a specific embodiment, the anti-KIT antibody is a naked antibody. In a specific embodiment, the anti-KIT antibody is a bivalent monospecific antibody. In a specific embodiment, the anti-KIT antibody is a bispecific antibody. In a certain embodiment, the anti-KIT antibody is not a bispecific antibody.

[0271] Table 4: VH amino acid sequences

[0272]

[0273] Table 5: VL amino acid sequences

[0274]

[0275] In a specific aspect, the anti-KIT antibodies (e.g., humanized antibodies) provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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.

[0276] In one embodiment, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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.

[0277] In one embodiment, the anti-KIT antibodies provided herein comprise 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 antibodies provided herein comprise 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 antibodies provided herein comprise 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 antibodies provided herein comprise 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: 15. In one embodiment, the anti-KIT antibodies provided herein comprise 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.

[0278] In one embodiment, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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.

[0279] In one embodiment, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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, the anti-KIT antibodies provided herein comprise 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.

[0280] In a specific aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises:

[0281] (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

[0282] (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.

[0283] It has been previously found that anti-KIT antibodies can induce degranulation of human mast cells expressing FcgRI and / or exhibit Fc receptor-dependent KIT agonist activity, which may lead to undesirable infusion-related reactions (IRRs) and other adverse effects.

[0284] In various embodiments, the anti-KIT antibodies or antigen-binding fragments described herein comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (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 human mast cells expressing FcgRI, and / or exhibit Fc receptor-dependent KIT agonist activity. In certain embodiments, one or more of these properties of the anti-KIT antibody or antigen-binding fragment is due to the modified (e.g., mutated) Fc region or domain.

[0285] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein have reduced Fc receptor binding activity (particularly reduced FcγR binding activity). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not have significant Fc receptor (particularly FcγR) binding activity. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not have detectable Fc receptor (particularly FcγR) binding activity. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein have 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 an appropriate control antibody or antigen-binding fragment. When the anti-KIT antibodies or antigen-binding fragments described herein comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain), in preferred embodiments, an appropriate 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, the anti-KIT antibodies or antigen-binding fragments described herein comprise a modified (e.g., mutated) human IgG1 Fc region or domain and have Fc receptor (particularly FcγR) binding activity that 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 that of a corresponding antibody or antigen-binding fragment with the same VH and VL but with a wild-type (unmodified) human IgG1 Fc region or domain.

[0286] 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, for example, the percent release of β-hexosaminidase in human mast cells cultured (e.g., in the presence of IFNγ)). 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, for example, the percent release of β-hexosaminidase in human mast cells cultured (e.g., in the presence of IFNγ)). In particular embodiments, the anti-KIT antibodies or antigen-binding fragments described herein induce at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less degranulation of human mast cells expressing FcgRI (e.g., as determined by, for example, the percent release of β-hexosaminidase in human mast cells cultured (e.g., in the presence of IFNγ)) compared to an appropriate control antibody or antigen-binding fragment. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein reduce the amount of β-hexosaminidase released from human mast cells cultured in the presence of IFNγ by more than 50%, compared to an appropriate control antibody or antigen-binding fragment. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein reduce the amount of β-hexosaminidase released from human mast cells cultured in the presence of IFNγ by more than 60%, more than 70%, or more than 80%, compared to an appropriate control antibody or antigen-binding fragment. When the anti-KIT antibodies or antigen-binding fragments described herein comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain), in preferred embodiments, the appropriate control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL but having 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%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% less degranulation in FcgRI-expressing human mast cells (e.g., as determined by the percentage release of β-hexosaminidase in human mast cells in culture (e.g., in the presence of IFNγ)) 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 amount of β-hexosaminidase released from human mast cells cultured in the presence of IFNγ is reduced by greater than 50% using an anti-KIT antibody or antigen-binding fragment comprising a modified (e.g., mutated) human IgG1 Fc region or domain as described herein, 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 amount of β-hexosaminidase released from human mast cells cultured in the presence of IFNγ is reduced by greater than 60%, greater than 70%, or greater than 80% using an anti-KIT antibody or antigen-binding fragment comprising a modified (e.g., mutated) human IgG1 Fc region or domain as described herein, 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.

[0287] 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, for example, 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, for example, KIT phosphorylation). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein induce Fc receptor-dependent KIT activity (e.g., as determined by, for example, KIT phosphorylation) that 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 compared to an appropriate control antibody or antigen-binding fragment. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein reduce Fc receptor-dependent KIT agonist activity (as determined by Fc receptor-cross-linked KIT phosphorylation) by more than 50% compared to an appropriate control antibody or antigen-binding fragment. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein reduce Fc receptor-dependent KIT agonist activity (determined by Fc receptor-cross-linked KIT phosphorylation) by greater than 60%, greater than 70%, or greater than 80% compared to an appropriate control antibody or antigen-binding fragment. When the anti-KIT antibodies or antigen-binding fragments described herein comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (e.g., mutated) human IgG1, IgG2, IgG3, or IgG4 Fc region or domain), in preferred embodiments, the appropriate control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL but having 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 Fc receptor-dependent KIT activity (e.g., as determined by, for example, KIT phosphorylation) that 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 the same VH and VL but having a wild-type (unmodified) human IgG1 Fc region or domain. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not exhibit significant or detectable Fc receptor-dependent KIT agonist activity as described herein even when cross-linked on THP-1 cells.In certain embodiments, the Fc receptor-dependent KIT agonist activity (determined by Fc receptor-crosslinked KIT phosphorylation) is reduced by more than 50% using an anti-KIT antibody or antigen-binding fragment described herein comprising 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 Fc receptor-dependent KIT agonist activity (determined by Fc receptor-crosslinked KIT phosphorylation) is reduced by more than 60%, more than 70%, or more than 80% using an anti-KIT antibody or antigen-binding fragment described herein comprising 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.

[0288] In various embodiments, the anti-KIT antibodies or antigen-binding fragments described herein (1) reduce disease activity in CIndU patients (e.g., CIndU patients refractory to antihistamine therapy); (2) reduce the number of skin mast cells in CIndU patients (e.g., CIndU patients refractory to antihistamine therapy); (3) reduce tryptase levels in CIndU patients (e.g., CIndU patients refractory to antihistamine therapy); (4) improve the clinical course of CIndU patients (e.g., (5) improving the quality of life of CIndU patients (e.g., CIndU patients refractory to antihistamine therapy); and / or (6) maintaining hematological parameters (e.g., hemoglobin (HgB) levels, white blood cell (WBC) counts, platelet counts, and / or absolute neutrophil counts (ANC)) in patients, such as CIndU patients (e.g., CIndU patients refractory to antihistamine therapy) within normal ranges.

[0289] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly reduce CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) in patients with CIndU, relative to pre-treatment values. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can increase the sensitivity of CIndU patients (e.g., CIndU patients whose CIndU are refractory to antihistamine therapy) to a critical temperature threshold in the range of 0.04 to 0.1%. In some embodiments, the critical temperature threshold in the treatment arm is reduced 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 one, two, four, six, eight, ten, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In certain embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists for at least two, four, six, eight, ten, or twelve weeks.

[0290] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly reduce the number of injections in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) relative to the number of injections before treatment. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can allow CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) to receive at least one injection of KIT in the presence of a single injection, relative to the number of injections prior to treatment. In certain embodiments, the number of injections in a patient's blood is reduced by at least 1, at least 2, at least 3, or at least 4 (e.g., within one, two, four, six, eight, ten, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In certain embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists for at least two, at least four, at least six, at least eight, at least ten, or at least twelve weeks.

[0291] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly improve Physician's Global Assessment (Phys-GA) and / or Patient's Global Assessment (Pat-GA), relative to pre-treatment levels. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can improve Physician's Global Assessment (Phys-GA) and / or Patient's Global Assessment (Pat-GA), by reducing the Physician's Global Assessment (Phys-GA) and / or Patient's Global Assessment (Pat-GA) on a Likert scale (0-3, 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, relative to pre-treatment levels (e.g., within one, two, four, six, eight, ten, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists 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.

[0292] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly reduce the number of skin mast cells in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) relative to the number before treatment. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can reduce the number of skin mast cells in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) by at least 20%, at least 40%, at least 60%, or at least 80% relative to the number before treatment (e.g., within one week, two weeks, four weeks, six weeks, eight weeks, ten weeks, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists for at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, or at least twelve weeks.

[0293] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly reduce serum tryptase in CIndU patients (e.g., CIndU patients whose CIndU are refractory to antihistamine therapy) relative to pre-treatment levels. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can reduce serum tryptase in CIndU patients (e.g., CIndU patients whose CIndU are refractory to antihistamine therapy) by at least 50%, at least 70%, or at least 90% relative to pre-treatment levels (e.g., within one, two, four, six, eight, ten, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists for at least two, at least four, at least six, at least eight, at least ten, or at least twelve weeks.

[0294] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly improve the control of urticaria in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy), relative to pre-treatment levels. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can improve the control of urticaria in CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) by increasing the Urticaria Control Test (UCT) score by at least 2 points, at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, at least 11 points, at least 12 points, at least 13 points, at least 14 points, at least 15 points, or 16 points relative to pre-treatment levels, or by increasing the UCT score to at least 12 points, at least 13 points, at least 14 points, at least 15 points, or 16 points (e.g., within one, two, four, six, eight, ten, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists 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.

[0295] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can significantly improve the quality of life of CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy), relative to pre-treatment levels. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments described herein can improve the quality of life of CIndU patients (e.g., CIndU patients whose CIndU is refractory to antihistamine therapy) by increasing the Dermatology Life Quality Index (DLQI) by at least 2 points, at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, at least 12 points, at least 14 points, at least 16 points, at least 18 points, at least 20 points, or at least 25 points, relative to pre-treatment levels, or by reducing the DLQI by at most 5 points, at most 4 points, at most 3 points, at most 2 points, at most 1 point, or 0 points (e.g., within one, two, four, six, eight, ten, or twelve weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In specific embodiments, the effect of the anti-KIT antibody or antigen-binding fragment persists 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.

[0296] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein maintain a patient's hematological parameters (e.g., hemoglobin (HgB) levels, white blood cell (WBC) counts, platelet counts, and / or absolute neutrophil counts (ANC)) within a normal range. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments described herein maintain a patient's hematological parameters (e.g., hemoglobin (HgB) levels, white blood cell (WBC) counts, platelet counts, and / or absolute neutrophil counts (ANC)) within a normal range in CIndU patients (e.g., CIndU patients whose CIndU are refractory to antihistamine therapy). In specific embodiments, the hematological parameters are maintained 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.

[0297] In various embodiments, the anti-KIT antibodies described herein have one or more properties described herein. In various embodiments, the antigen-binding fragments of the anti-KIT antibodies described herein have one or more properties described herein.

[0298] In specific embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein said Fc region or domain comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., one, two, three, four, five, or six) non-naturally occurring amino acid residue.

[0299] In a specific embodiment, an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is that of human IgG1 and comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., one, two, three, four, five, or six) non-naturally occurring amino acid residue, numbered according to the EU index as set forth in Kabat,The amino acid residue is selected from the group consisting of 234A, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 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, 241 L, 241Y, 241E, 241R.243W, 243L243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 2 63T, 263M, 264L, 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, 296H, 269G, 297S, 297D, 2 97E, 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, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A. Optionally,The Fc region or domain may comprise additional and / or alternative non-naturally occurring amino acid residues known to those skilled in the art (see, e.g., U.S. Patents 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 05 / 040217). In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG2 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modification (e.g., substitution, deletion or addition) or at least one (e.g., one, two, three, four, five or six) non-naturally occurring amino acid residue that is equivalent to the amino acid residues of the human IgG1 Fc region or domain described herein, which can be determined by one skilled in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG3 and comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., one, two, three, four, five, or six) non-naturally occurring amino acid residue, which is equivalent to the amino acid residues of a human IgG1 Fc region or domain described herein, as can be determined by one skilled in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG4 and comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., one, two, three, four, five, or six) non-naturally occurring amino acid residue, which is equivalent to the amino acid residues of a human IgG1 Fc region or domain described herein, as can be determined by one skilled in the art.

[0300] In a specific embodiment, an antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain is that of human IgG1 and comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one non-naturally occurring amino acid residue (e.g., one, two, three, four, five, or six), numbered according to the EU index as set forth in Kabat,The amino acid residue is selected from the group consisting of 234A, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 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, 241 L, 241Y, 241E, 241R.243W, 243L243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 2 63T, 263M, 264L, 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, 296H, 269G, 297S, 297D, 2 97E, 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, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A. Optionally,The Fc region or domain may comprise additional and / or alternative non-naturally occurring amino acid residues known to those skilled in the art (see, e.g., U.S. Patents 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 05 / 040217). In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG2 and comprises at least one (e.g., one, two, three, four, five or six) amino acid modification (e.g., substitution, deletion or addition) or at least one (e.g., one, two, three, four, five or six) non-naturally occurring amino acid residue that is equivalent to the amino acid residues of the human IgG1 Fc region or domain described herein, which can be determined by one skilled in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG3 and comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., one, two, three, four, five, or six) non-naturally occurring amino acid residue, which is equivalent to the amino acid residues of a human IgG1 Fc region or domain described herein, as can be determined by one skilled in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG4 and comprises at least one (e.g., one, two, three, four, five, or six) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., one, two, three, four, five, or six) non-naturally occurring amino acid residue, which is equivalent to the amino acid residues of a human IgG1 Fc region or domain described herein, as can be determined by one skilled in the art.

[0301] In one aspect, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is that of human IgG1 and comprises at least one non-naturally occurring amino acid, numbered according to the EU index as set forth in Kabat, at one or more positions selected from the group consisting of 239, 330, and 332. In a specific embodiment, provided herein is an antibody comprising an Fc region or domain, wherein the Fc region or domain is that of human IgG1 and comprises at least one non-naturally occurring amino acid, numbered according to the EU index as set forth in Kabat, at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L, and 332E. Optionally, the Fc region or domain may further comprise an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, numbered according to 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 an Fc region or domain of human IgG1 and comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L, and 332E, according to the EU index numbering as set forth in Kabat, and at least one non-naturally occurring amino acid at one or more positions is selected from the group consisting of 252Y, 254T, and 256E, according to the EU index numbering 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 an Fc region or domain of human IgG2, IgG3, or IgG4, and comprises at least one non-naturally occurring amino acid residue that is equivalent to an amino acid residue of a human IgG1 Fc region or domain described herein, which can be determined by one skilled in the art. In a specific embodiment, an antibody comprising an Fc region or domain is provided herein, wherein the Fc region or domain is an Fc region or domain of human IgG2, IgG3 or IgG4, and comprises at least one non-naturally occurring amino acid residue located at one or more positions, and the one or more positions are comparable to the positions of the human IgG1 Fc region or domain described herein, which can be determined by those skilled in the art. In one embodiment, the Fc region or domain comprising such a sequence exhibits one or more Fc activities, such as binding affinity or effector functions to an Fc receptor, such as ADCC or CDC. In a specific embodiment, the Fc region or domain comprising such a sequence exhibits reduced Fc activity, such as reduced binding affinity to an Fc receptor or reduced effector functions, such as ADCC or CDC. In a specific embodiment, the Fc region or domain comprising such a sequence exhibits enhanced FcRn activity, such as enhanced half-life.

[0302] Additional non-limiting examples of Fc region or domain modifications are provided 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 J9:115-119; 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 82:57-65cPresta 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; ,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 No. 2004 / 0002587 and PCT Publication Nos. WO 94 / 29351; WO 99 / 58572; WO 00 / 42072; WO 04 / 029207; WO 04 / 099249: WO 04 / 063351. .

[0303] In specific embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q, and 322Q, according to the EU index numbering as set forth in Kabat. In a specific embodiment, the modified (e.g., mutated) human IgG1 Fc region or domain further comprises the non-naturally occurring amino acids 252Y, 254T, and 256E, according to the EU index numbering as set forth in Kabat.

[0304] In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG2 Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, and 322Q as numbered by the EU index as set forth in Kabat for a human IgG1 Fc region or domain, as can be determined by one skilled in the art. In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG2 Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E as numbered by the EU index as set forth in Kabat for a human IgG1 Fc region or domain, as can be determined by one skilled in the art.

[0305] In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG3 Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, and 322Q as numbered by the EU index as set forth in Kabat for a human IgG1 Fc region or domain, as can be determined by one skilled in the art. In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG3 Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E as numbered by the EU index as set forth in Kabat for a human IgG1 Fc region or domain, as can be determined by one skilled in the art.

[0306] In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG4 Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, and 322Q as numbered by the EU index as set forth in Kabat for a human IgG1 Fc region or domain, as can be determined by one skilled in the art. In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG4 Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E as numbered by the EU index as set forth in Kabat for a human IgG1 Fc region or domain, as can be determined by one skilled in the art.

[0307] In a specific embodiment, an antibody described herein comprises the VL and VH CDR sequences shown in Table 1 and a modified (e.g., mutated) human IgG1 Fc region or domain, wherein the modified (e.g., mutated) human IgG1 Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q, and 322Q according to the EU index numbering as set forth in Kabat.

[0308] In a preferred embodiment, the antibodies described herein comprise the VL and VH CDR sequences shown in Table 1 and a modified (e.g., mutated) human IgG1 Fc region or domain, wherein the modified (e.g., mutated) human IgG1 Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E according to the EU index numbering as set forth in Kabat.

[0309] Thus, in one aspect, provided herein is an antibody that immunospecifically binds to human KIT, the antibody comprising:

[0310] (i) a VL comprising VLCDR1, VLCDR2, and VLCDR3, wherein the VLCDR1, VLCDR2, and VL CDR3 have the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; (ii) a VH comprising VHCDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 have 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 non-naturally occurring amino acids 234A, 235Q, and 322Q according to the EU index numbering as set forth in Kabat.

[0311] Thus, in another aspect, provided herein is an antibody that immunospecifically binds to human KIT, the antibody comprising:

[0312] (i) a VL comprising VLCDR1, VLCDR2, and VLCDR3, wherein the VLCDR1, VLCDR2, and VL CDR3 have the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; (ii) a VH comprising VHCDR1, VH CDR2, and VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 have 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 non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E according to the EU index numbering as set forth in Kabat.

[0313] In another aspect, provided herein is an antibody that immunospecifically binds to human KIT, the antibody comprising: (i) a VL comprising the following amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X K1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 Is an amino acid with an aliphatic hydroxyl side chain or P, X K5 is an amino acid with a charged or acidic side chain, and X K6 is an amino acid having an aromatic side chain; and (ii) a VH comprising the following amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAPGKGLEWIARIYPGSGNTYYNEKFKGRX H4 TX H5 TAX H6 KSTSTAYMX HH7 LSSLRSEDX HH8AVYFCARGVYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, 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 comprising the non-naturally occurring amino acids 234A, 235Q, 322Q, and preferably further comprising 252Y, 254T, and 256E, according to the EU index numbering as set forth in Kabat.

[0314] In another aspect, provided herein is an antibody that immunospecifically binds to human KIT, the antibody 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 comprising the non-naturally occurring amino acids 234A, 235Q, 322Q, and preferably further comprising 252Y, 254T, and 256E, according to the EU index numbering as set forth in Kabat.

[0315] In another aspect, provided herein is an antibody that immunospecifically binds to human KIT, the antibody 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 comprising the non-naturally occurring amino acids 234A, 235Q, 322Q, and preferably further comprising 252Y, 254T, and 256E, according to the EU index numbering as set forth in Kabat.

[0316] In specific embodiments, the antibodies provided herein comprise a heavy chain comprising the following amino acid sequence:

[0317]

[0318] In specific embodiments, an antibody provided herein comprises a light chain comprising the following amino acid sequence:

[0319]

[0320] In specific embodiments, the antibodies provided herein comprise: a heavy chain comprising the following amino acid sequence:

[0321]

[0322] and a light chain comprising the following amino acid sequence:

[0323]

[0324] In a specific embodiment, provided herein is an antibody comprising: (i) a heavy chain comprising the following amino acid sequence: Wherein the leader sequence is shown in bold italics, the variable region (VH) is shown in italics and the constant region is shown underlined. In addition, the mutations in the constant region (compared to wild-type human IgG1) are shown in double underline; and

[0325] (ii) a light chain comprising the following amino acid sequence: The leader sequence is shown in bold italics, the variable region (VL) is shown in italics and the constant region is underlined.

[0326] In a specific embodiment, the anti-KIT antibodies described herein do not bind to any human Fc-gamma receptor (FcγR receptor) (e.g., no detectable binding). In a specific embodiment, the anti-KIT antibodies described herein do not bind to human FcγRI (e.g., no detectable binding). In a specific embodiment, the anti-KIT antibodies described herein do not bind to human FcγRIIa (e.g., no detectable binding). In a specific embodiment, the anti-KIT antibodies described herein do not bind to human FcγRIIb (e.g., no detectable binding). In a specific embodiment, the anti-KIT antibodies described herein do not bind to human FcγRIIIa (e.g., no detectable binding). In a specific embodiment, the anti-KIT antibodies described herein do not bind to human FcγRIIIb (e.g., no detectable binding).

[0327] In specific embodiments, 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 have enhanced binding (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, or 10,000-fold greater binding affinity) to the human neonatal Fc receptor (FcRn) relative 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 antibodies described herein bind to FcRn with a KD of less than 20 nM at pH 6.0. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 2 nM at pH 6.0. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 1 nM at pH 6.0. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 500 nM at pH 6.0. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 400 pM at pH 6.0. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 200 nM at pH 7.2. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 150 nM at pH 7.2. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 100 nM at pH 7.2. In a specific embodiment, the anti-KIT antibodies described herein bind to FcRn with a KD of less than 80 nM at pH 7.2.

[0328] In specific embodiments, 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 specific embodiments, 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% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or at least 99% lower) relative to a corresponding antibody having the same variable region sequence but an unmodified (wild-type) human IgG constant region.

[0329] In specific embodiments, 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 decreased (e.g., at least 10% less, at least 20% less, 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) cytokine (e.g., IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-10, and / or TNF-α) production relative to a corresponding antibody having the same variable region sequence but an unmodified (wild-type) human IgG constant region.

[0330] In certain aspects, antigen-binding fragments of the antibodies described herein are also provided, preferably antigen-binding fragments comprising a full-length heavy chain Fc region or domain (e.g., full-length human IgG1, human IgG2, human IgG3, or human IgG4 Fc region or domain). In certain aspects, antigen-binding fragments of the antibodies described herein are also provided, comprising a partial heavy chain Fc region or domain (e.g., a partial human IgG1, human IgG2, human IgG3, or human IgG4 Fc region or domain).

[0331] In certain aspects, anti-KIT antibodies or antigen-binding fragments thereof can be obtained using methods known in the art, e.g., see Section 5.4, below.

[0332] In one specific aspect, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the D4 domain of human KIT (e.g., human KIT) and the D5 region of KIT. In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the D5 domain of KIT (e.g., human KIT) with an affinity that is lower than the affinity with which it binds to the D4 domain of KIT (e.g., human KIT). In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the D4 domain of KIT (e.g., human KIT) with an affinity that is higher than the affinity with which it binds 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, such as ELISA or Biacore assays.

[0333] In a specific embodiment, 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 the D4 domain alone that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold greater than the affinity for a KIT antigen consisting essentially of the D5 domain alone.

[0334] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to a KIT polypeptide (e.g., D4 region of human KIT) as determined by an assay described in the art (e.g., ELISA) with an EC 50 (half maximal effective concentration) value is about 50 nM, 10 nM, 500 pM, 300 pM, 200 pM, 100 pM or 50 pM or less.

[0335] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to a KIT polypeptide (e.g., the D4 region of human KIT) with an EC50 value of about 200 pM or 150 pM or less as determined by an assay described in the art, such as ELISA or FAC using CHO-WT-KIT cells (CHO cells engineered to recombinantly express wild-type human KIT).

[0336] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of blocking KIT phosphorylation with an IC50 (50% inhibitory concentration) value of about 600 pM or less.

[0337] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing 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%, relative 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 one of skill in the art. In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing KIT receptor internalization, e.g., by at least about 25% or 35%, optionally by about 75%, relative 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 one of skill in the art. In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing KIT receptor internalization relative to internalization in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind), as assessed by methods described herein or known to those skilled in the art, e.g., inducing or enhancing 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. Techniques for quantifying or visualizing cell surface receptors are well known in the art and include a variety of 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 radioactive label and incubated with the cells. Additional receptor internalization assays are well 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.

[0338] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing 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%, relative to the turnover rate 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 (e.g., a pulse-chase assay). In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing KIT receptor turnover by at least about 25% or 35%, optionally to about 75%, relative to the turnover rate 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 (e.g., a pulse-chase assay). In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing 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 relative to turnover in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind) as assessed by methods described herein or known to those skilled in the art (e.g., a pulse-chase assay). Methods for determining receptor turnover are well known in the art. For example, one can use 35 KIT-expressing cells are pulse-labeled with S-EXPRESS protein labeling mix (NEG772, NEN Life Science Products), washed with unlabeled medium and chased for a period of time, and then protein lysates from the labeled cells are immunoprecipitated using anti-KIT antibodies and resolved and visualized by SDS-PAGE (e.g., exposed to a PhosphoImager screen (Molecular Dynamics), scanned using a Typhoon 8600 scanner (Amersham), and analyzed using ImageQuant software (Molecular Dynamics)) (see, e.g., Chan et al., Development, 2004, 131: 5551-5560).

[0339] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing degradation of the KIT receptor 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% relative 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 (e.g., a pulse-chase assay). In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing degradation of the KIT receptor by at least about 25% or 35%, optionally to about 75%, relative 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 (e.g., a pulse-chase assay). In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are capable of inducing or enhancing degradation of the KIT receptor 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 relative to degradation in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind) as assessed by methods described herein or known to those skilled in the art (e.g., pulse-chase assays). Techniques for quantifying or monitoring ubiquitination and / or degradation (e.g., degradation kinetics or rates) of cell surface receptors are well known in the art and involve a variety of fluorescent and radioactive techniques (see, e.g., International Patent Application Publication No. WO2008 / 153926 A2). For example, pulse-chase experiments can be performed or radiolabeled ligands (e.g., 125 I-SCF) experiments were performed to quantitatively measure KIT degradation.

[0340] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein do not bind to the extracellular ligand binding site of KIT, e.g., the SCF binding site of KIT. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein do not inhibit ligand binding to KIT, e.g., do not inhibit binding of a KIT ligand (e.g., SCF) to KIT, as determined by methods described in the art, e.g., ELISA. In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein do not completely inhibit, or partially inhibit, ligand binding to KIT, e.g., do not completely inhibit, or partially inhibit binding of a KIT ligand (e.g., SCF) to KIT, as determined by methods described in the art, e.g., ELISA or FACS (fluorescence activated cell sorting).

[0341] 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 a specific embodiment, partial inhibition of KIT activity results in, for example, about 25% to about 65% or 75% inhibition. In a specific embodiment, partial inhibition of KIT activity results in, for example, about 35% to about 85% or 95% inhibition. Non-limiting examples of KIT activity include KIT dimerization, KIT phosphorylation (e.g., tyrosine phosphorylation), KIT downstream signaling (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 specific embodiments, the antibodies described herein inhibit KIT phosphorylation (e.g., ligand-induced phosphorylation).

[0342] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT tyrosine phosphorylation in the KIT cytoplasmic domain.

[0343] In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit cell proliferation, such as mast cell proliferation or eosinophil proliferation. In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit cell survival, such as mast cell survival or eosinophil survival. In certain aspects, 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%.

[0344] In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit mast cell activation or eosinophil activation. In certain aspects, the 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%.

[0345] In a specific embodiment, 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 certain aspects, the inhibition of eosinophil or mast cell degranulation is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0346] In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit the release of mast cell mediators. In some aspects, mast cell mediator release is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. Described herein are assays for measuring mast cell activity, such as mast cell cultures, such as rodent and human mast cell culture mediator release (see, for example, 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.)). For example, certain assays are designed to monitor mast cell degranulation by measuring the release of granule components, such as β-hexosaminidase, determining the production of phospholipid metabolites such as eicosanoids, leukotriene C4 (LTC4) and prostaglandin D2 (PGD2), or determining the production of multiple cytokines. In certain aspects, enzyme-linked immunosorbent assays (ELISAs) can be used to measure cytokine release in mast cell cultures. In certain aspects, 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 determine the effect of anti-KIT antibodies on mast cells.

[0347] In one embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein induce cell apoptosis, such as mast cell apoptosis or eosinophil apoptosis. In another embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein induce cell differentiation, such as mast cell differentiation.

[0348] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein can achieve any of the following: reducing the number and / or activity of eosinophils, reducing mast cell proliferation, reducing plasma tryptase levels, reducing plasma SCF levels, reducing the number or amount of mast cells, inhibiting or reducing mast cell activity, reducing the production or release of inflammatory factors induced by mast cells, reducing the release of inflammatory factors, restoring mast cell homeostasis, reducing mast cell migration, reducing mast cell adhesion, inhibiting or reducing the recruitment of eosinophils by mast cells, and inhibiting or reducing antigen-mediated mast cell degranulation.

[0349] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity but do not inhibit KIT dimerization. In another specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity and do not inhibit ligand binding to KIT, e.g., do not inhibit binding of a KIT ligand (e.g., SCF) to KIT, but do inhibit KIT dimerization.

[0350] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity, e.g., ligand-induced tyrosine phosphorylation of the cytoplasmic domain of KIT, by about 25% to about 65% or 75%, as determined by a cell-based phosphorylation assay well known in the art, such as the cell-based phosphorylation assays described herein. In a certain embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity, e.g., ligand-induced tyrosine phosphorylation of the cytoplasmic domain of KIT, by about 35% to about 85% or 95%, as determined by a cell-based phosphorylation assay well known in the art, such as the cell-based phosphorylation assays described herein.

[0351] In a specific embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein inhibit KIT activity, such as ligand-induced tyrosine phosphorylation of the KIT cytoplasmic domain, at a 50% inhibitory concentration (IC 50 ) is less than about 600 pM, or less than about 500 pM, or less than about 250 pM, as determined by cell-based phosphorylation assays well known in the art, such as those described herein. In a specific embodiment, IC 50 Less than about 550 pM or 200 pM. In a specific embodiment, IC 50 In the range of about 50 pM to about 225 pM, or in the range of 100 pM to about 600 pM. In a specific embodiment, IC 50In the range of about 50 pM to about 550 pM, or in the range of about 50 pM to about 600 pM, or in the range of about 150 pM to about 550 pM.

[0352] In one embodiment, the anti-KIT antibodies or antigen-binding fragments thereof provided herein (i) immunospecifically bind to a KIT polypeptide comprising the D4 and / or D5 regions of human KIT; (ii) inhibit KIT phosphorylation (e.g., tyrosine phosphorylation); and (iii) incompletely inhibit, or partially inhibit, the binding of a KIT ligand (e.g., SCF) to KIT. In another embodiment, such an antibody does not inhibit KIT dimerization. In yet another embodiment, such an antibody can be recombinantly expressed in CHO cells with an average titer of at least 0.5 μg / mL, e.g., at least 1.0 μg / mL. In yet another embodiment, such an antibody comprises a non-immunogenic VH domain and a VL domain, e.g., a VH domain and a VL domain that do not contain T cell epitopes.

[0353] In other specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein immunospecifically bind to a monomeric form of KIT (e.g., human KIT). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein immunospecifically bind to a monomeric form of KIT (e.g., human KIT). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein immunospecifically bind to a dimeric form of KIT (e.g., human KIT).

[0354] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein do not bind to monomeric KIT and specifically bind to dimeric or multimeric KIT. In certain embodiments, the antibodies have a higher affinity for KIT monomers than for KIT dimers. In certain embodiments, the antibodies have a higher affinity for KIT monomers than for KIT multimers.

[0355] In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to a naturally occurring isoform or naturally occurring variant of KIT (i.e., a KIT isoform or variant naturally occurring 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 human)). In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof. In specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof and do 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 specific embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to human KIT or a fragment thereof and do not specifically bind to murine 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) as well as 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) as well as canine (dog) and non-human primate (e.g., monkey) KIT, but do not specifically bind to murine or rat KIT or a fragment thereof (e.g., the D4 region of murine KIT).

[0356] 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) as well as canine (dog), feline (cat), and cynomolgus monkey KIT, but do not specifically bind to murine or rat KIT or a fragment thereof (e.g., the D4 region of murine KIT).

[0357] 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), as well as canine (dog), feline (cat), and cynomolgus monkey KIT with an affinity that is higher (e.g., at least 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold) than to murine or rat KIT or a fragment thereof (e.g., the D4 region of murine KIT).

[0358] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the extracellular domain of human KIT comprising a mutation, e.g., a somatic mutation, such as a mutation in exon 9 of human KIT in which the Ala and Tyr residues at positions 502 and 503 are duplicated (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, each of which is incorporated herein by reference in its entirety, describing KIT mutations).

[0359] In certain embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein specifically bind to the glycosylated extracellular domain of human KIT. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to two differently glycosylated forms of the extracellular domain of human KIT. For example, two different molecular weights of human KIT are observed by immunoblotting, indicating different glycosylation patterns.

[0360] In certain embodiments, the antibodies described herein can specifically bind to two forms of human KIT that have different glycosylation patterns, e.g., one form is more highly glycosylated than the other form. In certain embodiments, the antibodies described herein or antigen-binding fragments thereof bind to the unglycosylated extracellular domain of human KIT.

[0361] In a specific embodiment, the anti-KIT antibody or antigen-binding fragment thereof provided herein is a bivalent monospecific antibody because it has 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 region comprises the VH and VL CDRs shown in Table 1. In a specific embodiment, the antigen-binding region comprises 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 antibody or antigen-binding fragment thereof provided herein is not a bispecific antibody.

[0362] In one embodiment, the antibodies described herein are monoclonal antibodies or isolated monoclonal antibodies. In another embodiment, the antibodies described herein are humanized monoclonal antibodies. In a specific embodiment, the antibodies described herein are recombinant antibodies, such as recombinant human antibodies, recombinant humanized antibodies, or recombinant monoclonal antibodies. In certain embodiments, the antibodies described herein contain non-human amino acid sequences, such as non-human CDRs or non-human (e.g., non-human primate) framework residues.

[0363] In specific embodiments provided herein, recombinant antibodies can be isolated, prepared, expressed, or produced recombinantly, such as 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 involving production, such as through synthesis, genetic engineering of DNA sequences encoding human immunoglobulin sequences, or splicing sequences encoding human immunoglobulins (e.g., human immunoglobulin gene sequences) to other such sequences. In certain embodiments, the amino acid sequence of such recombinant antibodies has been modified such that the amino acid sequence of such antibodies (e.g., VH and / or VL regions) is a sequence that does not naturally occur within an antibody germline repertoire (e.g., a mouse or human germline repertoire) in an organism. In a specific embodiment, a recombinant antibody can be obtained by assembling a composite sequence of a recombinant antibody from several sequence fragments that naturally occur in an organism (e.g., a primate, such as a human), wherein the composite sequence does not naturally occur in an organism (e.g., a primate, such as a human).

[0364] 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. In a specific embodiment, the antibodies provided herein are IgG antibodies (e.g., human IgG antibodies), or classes (e.g., human IgG1 or IgG4), or subclasses 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 intact antibodies, e.g., whole or intact humanized, human, or composite human antibodies.

[0365] In a specific aspect, the antibodies provided herein comprise antibody light chains and heavy chains, such as individual light chains and heavy chains. With regard to light chains, in a specific embodiment, the light chains of the antibodies described herein are kappa light chains. In another specific embodiment, the light chains of the antibodies described herein are lambda light chains. In yet another specific embodiment, the light chains of the antibodies described herein are human kappa light chains or human kappa light chains. In a specific embodiment, the antibodies described herein comprise human light chain constant regions. Non-limiting examples of human light chain constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services. NIH Publication No. 91-3242.

[0366] For heavy chains, in a specific embodiment, the heavy chains of the antibodies described herein can be α (alpha), δ (delta), ε (epsilon), γ (gamma) or μ (mu) heavy chains. In another specific embodiment, the heavy chains of the antibodies can comprise human α (alpha), δ (delta), ε (epsilon), γ (gamma) or μ (mu) heavy chains. In a specific embodiment, the antibodies described herein comprise human heavy chain constant regions (e.g., human IgG constant regions, such as human IgG1, IgG2, IgG3 or IgG4 constant regions). Non-limiting examples of human heavy chain constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. In a specific embodiment, an antibody described herein comprises 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).

[0367] In certain embodiments, the anti-KIT antibodies described herein are human, composite human, or humanized monoclonal antibodies. In a specific embodiment, the antibodies described herein are engineered antibodies, such as antibodies produced by recombinant methods. In a specific embodiment, 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 a specific embodiment, the antibodies described herein comprise one or more primate (or non-human primate) framework regions. In a specific embodiment, the antibodies described herein do not comprise non-human primate framework regions.

[0368] The antibodies provided herein can include antibodies containing chemical modifications, such as antibodies that have been chemically modified, for example, by covalently attaching any type of molecule to the antibody. For example, but not limited to, anti-KIT antibodies can be glycosylated, acetylated, pegylated, phosphorylated, or amidated; can be derivatized via protecting / blocking groups; or can additionally contain cellular ligands and or other proteins or peptides (e.g., heterologous proteins or peptides), etc. For example, the antibodies provided herein can be chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization using known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. In addition, the anti-KIT antibodies described herein can contain one or more non-classical amino acids.

[0369] In one embodiment, the anti-KIT antibodies provided herein are naked antibodies that are not linked, fused, or conjugated (e.g., artificially linked, fused, or conjugated) to another molecule, peptide, or polypeptide (e.g., a heterologous polypeptide). In a specific embodiment, the anti-KIT antibodies provided herein are not antibody-drug conjugates. In a specific embodiment, the anti-KIT antibodies provided herein are not fusion proteins. In a specific embodiment, the anti-KIT antibodies described herein do not contain any non-classical amino acids.

[0370] 5.1.1 Antibody Conjugates

[0371] In some embodiments, provided herein are antibodies (e.g., human or humanized antibodies) or antigen-binding fragments thereof that are conjugated or recombinantly fused to a diagnostic agent, a detectable agent, or a therapeutic agent, or any other molecule. The conjugated or recombinantly fused antibodies can be used, for example, to monitor or predict the occurrence, development, progression, and / or severity of a KIT-related disorder or disease, for example, as part of a clinical testing program, for example, to determine the efficacy of a particular therapy. The conjugated or recombinantly fused antibodies can be used, for example, to prevent, treat, or manage a KIT-related disorder, or to prevent, treat, or manage the effects of a KIT-related disorder. The antibodies described herein can also be conjugated to a molecule (e.g., polyethylene glycol) that can affect one or more biological and / or molecular properties of the antibody, such as stability (e.g., stability in serum), half-life, solubility, and antigenicity.

[0372] In a specific aspect, a conjugate is provided herein, comprising an agent (e.g., a therapeutic agent) attached to an antibody (or antigen-binding fragment thereof) described herein. In a specific embodiment, the conjugate comprises an antibody described herein and a molecule (e.g., a therapeutic or drug moiety), wherein the antibody is directly attached to the molecule, or is attached to the molecule through one or more linkers. In certain embodiments, the antibody is covalently conjugated to the molecule. In a specific embodiment, the antibody is non-covalently conjugated to the molecule. In a specific embodiment, the antibody described herein, e.g., an antibody conjugated to an agent, binds to wild-type human KIT. In certain embodiments, the antibody described herein, e.g., an antibody conjugated to an agent, is conjugated to the extracellular domain of human KIT comprising a mutation, e.g., a somatic mutation associated with cancer (e.g., GIST), e.g., a mutation in human KIT exon 9, wherein the Ala and Tyr residues at positions 502 and 503 are repeated.

[0373] Such diagnosis and detection can be achieved, for example, by coupling the antibody to a detectable molecule or substance, including but not limited to: 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 or avidin / biotin; fluorescent materials, such as but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to luminol; bioluminescent materials, such as but not limited to luciferase, luciferin or 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 (1 15 In, 113 In, 1 12 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 Such as 68 Ge, 57 Co、 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn and 117 Sn; and positron-emitting metals using various positron emission tomography methods; and non-radioactive paramagnetic metal ions.

[0374] 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, such as a cytostatic or cell-killing agent; a therapeutic agent, or a radioactive metal ion, such as an alpha emitter. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Therapeutic moieties include, but are not limited to, auristatins or derivatives thereof, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PYE, and auristatin E (AE) (see, e.g., 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); microtubule disrupting agents, such as maytansine or derivatives thereof, such as maytansine DM1 (see, e.g., U.S. Patent Nos. 7,851,432, 7,575,748, and 5,416,064, each of which is incorporated herein by reference); prodrugs, such as CC-1065 (rachelmycin); n)) analogs; antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine); alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C); C), cis-dichlorodiamine platinum (II) (DDP), and cisplatin); minor groove binding alkylating agents; anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (e.g., d-actinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)); auristatin molecules (e.g., auristatin PHE, bryostatin 1, and solastatin 10; see 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); hormones (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. 20:107-111), and imatinib mesylate (Kantarjian et al., Clin Cancer Res. 20:107-111). Res.8(7):2167-76(2002));Cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, anthracindione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or homologs, and U.S. Patent Nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271, those disclosed in 5,728,868, 5,648,239, 5,587,459, each of which is incorporated herein by reference for its disclosure of such compounds);Farnesyltransferase inhibitors (e.g., R115777, BMS-214662, and, for example, U.S. Pat. Nos. 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, 6,399,615, 6,387,90 5, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, ​​6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,1 93、6,187,786、6,169,096、6,159,984、6,143,766、6,133,303、6,127,366、6,124,465、6,124,295、6,103,723、6,093,737、6,090,948、6,080,870、6,077,853、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; saintopin; 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; coralyne; beta-lapachone; BC-4-1;Bisphosphonates (e.g., alendronate, cimadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolendronate) HMG-CoA reductase inhibitors (e.g., lovastatin, simvastatin) atin, atorvastatin, pravastatin, fluvastatin, statin, cerivastatin, lescol, lupitor, rosuvastatin, and atorvastatin); antisense oligonucleotides (e.g., those disclosed in U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709, each of which is incorporated herein by reference); adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetan; tositumomab ) and pharmaceutically acceptable salts, solvates, clathrates and prodrugs thereof.

[0375] In certain embodiments, the therapeutic moiety or drug moiety is an anti-tubulin drug, such as auristatin or a derivative thereof. Non-limiting examples of auristatin include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin PYE and auristatin E (AE) (see, e.g., 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 certain embodiments, the therapeutic moiety or drug moiety is a microtubule disrupting agent, such as maytansine or a derivative thereof, such as maytansine DM1 or DM4 (see, e.g., 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, such as 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).

[0376] In one embodiment, the antibody and therapeutic agent / agent are conjugated via one or more linkers. In another embodiment, the antibody and therapeutic agent / agent are directly conjugated.

[0377] In specific embodiments, non-limiting examples of therapeutic or drug moieties for conjugation to the antibodies described herein include calicheamicins (e.g., LL-E33288 complex, e.g., gamma-calicheamicin, see, e.g., U.S. Patent No. 4,970,198) and derivatives thereof (e.g., gamma-calicheamicin hydrazide derivatives), ozogamicins, duocarmycins and derivatives thereof (e.g., CC-1065 (NSC 298223) or achiral analogs of duocarmycin (e.g., AS-1-145 or centanamycin)), taxanes and derivatives thereof, and enediynes and derivatives thereof (see, e.g., PCT International Patent Application Publication Nos. WO 2009 / 017394, WO 2010 / 062171, WO 2007 / 089149, WO 2011 / 021146, WO 2008 / 150261, WO 2006 / 031653, WO 2005 / 089809, WO 2005 / 089807 and WO 2005 / 089808, each of which is incorporated herein by reference in its entirety).

[0378] Non-limiting examples of calicheamicins suitable for conjugation to the antibodies described herein are disclosed in, for example, U.S. Patent 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; the disclosures of each of these cases regarding calicheamicins are incorporated herein by reference. In certain embodiments, these compounds may contain methyl trisulfide, which reacts with an appropriate thiol to form a disulfide and simultaneously introduces a functional group such as a hydrazide or other functional group that can be used to conjugate calicheamicin to the antibodies described herein. In certain embodiments, improved antibody / drug conjugates can be produced by adding dimethyl substituents to stabilize the disulfide bonds present in the calicheamicin conjugate. In a specific embodiment, the calicheamicin derivative is N-acetyl gamma calicheamicin dimethyl hydrazide, or NAc-γDMH (CL-184,538), as one of the optimized derivatives for conjugation. For example, calicheamicin disulfide analogs that can be conjugated to the antibodies described herein are described 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 a certain embodiment, a portion (e.g., calicheamicin or a derivative thereof) is conjugated to the antibody via a linker. In a specific embodiment, a portion (e.g., calicheamicin or a derivative thereof) is hydrolyzed from the linker of the antibody-drug conjugate. In one embodiment, the moiety (eg, calicheamicin or a derivative thereof) is hydrolyzed from the linker of the antibody conjugate at a temperature between 20 and 50°C, preferably at 37°C, between about pH 3.0 and pH 4.0, for 1-24 hours.

[0379] 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), PBD dimer prodrugs activated by hydrolysis (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 2005 / 040170, and U.S. Pat. No. 7,612,062, the disclosures of which regarding such compounds are incorporated herein by reference).

[0380] Furthermore, the antibodies described herein can be conjugated to a therapeutic moiety, such as a radioactive metal ion, for example, an alpha emitter, e.g. 213 Bi, or can be used to convert radioactive metal ions (including but not limited to 131 In, 131 LU, 131 Y. 131 Ho, 131 Sm) macrocyclic chelators conjugated to polypeptides. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), which can be linked to the antibody via a linker molecule. Such linker molecules are well 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):553-7c and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

[0381] In certain embodiments, antibody described herein or its Fab is directly or indirectly conjugated to one or more molecules (such as treatment or drug moiety) through one or more linker molecules.In specific embodiments, joint is enzyme cleavable joint or disulfide joint.In a specific embodiment, cleavable joint can be cracked by enzyme such as aminopeptidase, aminoesterase, dipeptidyl carboxypeptidase or the protease of blood coagulation cascade.In specific embodiments, joint comprises 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 or 20 amino acid residues.In certain embodiments, joint is by 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.

[0382] In certain embodiments, part is conjugated to the antibody through one or more linkers. In a specific embodiment, part is hydrolyzed from the linker of the antibody-drug conjugate. In one embodiment, part is hydrolyzed from the linker of the antibody conjugate at a temperature of 20 to 50 ° C, preferably at a temperature of 37 ° C, between about pH 3.0 and pH 4.0, for about 1-24 hours. In a specific embodiment, the linker is stable in the bloodstream, but once it enters the target cell, the conjugated part will be released. In certain embodiments, part is conjugated to the antibody described herein through 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.

[0383] In addition, the antibodies provided herein can be fused to a tag sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the tag amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc.), many of which are commercially available. For example, as described by Gentz ​​et al. in Proc. Natl. Acad. Sci. USA 86:821-824 in 1989, hexahistidine will facilitate purification of the fusion protein. Other peptide tags that can be used 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.

[0384] Methods for fusing or conjugating therapeutic moieties, including polypeptides, to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in 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," in 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 Antibodies In Cancer Therapy," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); 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; 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 herein by reference in their entirety.

[0385] The antibodies described herein can also be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0386] In one aspect, the antibodies or antigen-binding fragments thereof described herein are extracellular drug conjugates (ECDs) comprising an antibody linked to a drug (optionally linked via a linker) (see, e.g., PCT International Patent Application Publication No. WO 2011 / 031870). The drug can act extracellularly and therefore does not require internalization of the conjugate. Once the ECD binds to a target cell, the drug sends a signal to the cell.

[0387] In one embodiment, the linker of the ECD is a non-cleavable linker. Examples of non-cleavable linkers include linkers containing polyethylene glycol chains or polyethylene chains, which are insensitive to acids or bases (e.g., linkers containing hydrazones), insensitive to reducing agents or oxidizing agents (e.g., linkers containing disulfide bonds), and insensitive to enzymes that may be present in cells or circulatory systems. Specific examples of non-cleavable linkers include SMCC linkers (U.S. Patent Application 20090202536). For illustrative purposes, examples of cleavable linkers include linkers containing non-hindered glutathione-sensitive disulfides, esters, peptide sequences that are sensitive to peptidases (e.g., cathepsin or plasmin), pH-sensitive hydrazones (see Bioconjugate Chem., 2010, 21 (1), pp. 5-13), and the non-hindered disulfide linker SPP (U.S. Patent Application 20090202536).

[0388] In certain aspects, the ECD comprises a cardiac glycoside drug or agent, such as proscillaridin or sugar-enhanced proscillaridin. In one embodiment, the agent is composed of a sugar-free cardiac glycoside. In various embodiments, the cardiac glycoside is a compound identified in PCT Publication No. WO 2010 / 017480 (PCT / US2009 / 053159).

[0389] 5.2 Polynucleotides

[0390] In certain aspects, provided herein are polynucleotides and combinations of polynucleotides comprising nucleotide sequences encoding antibodies (e.g., human or humanized antibodies) or fragments thereof (e.g., variable light chain regions and / or variable heavy chain regions) described herein that immunospecifically bind to a KIT antigen. Also provided herein are polynucleotides encoding a KIT antigen for use in generating anti-KIT antibodies described herein.

[0391] As used herein, an "isolated" polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., the human body). In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or can be substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the phrase "substantially free" includes polynucleotide or nucleic acid molecule preparations having less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (particularly less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals. In a specific embodiment, one or more nucleic acid molecules encoding the antibodies described herein are isolated or purified.

[0392] In a specific aspect, provided herein are polynucleotides or combinations of polynucleotides comprising nucleotide sequences encoding an antibody or antigen-binding fragment thereof described herein, or the VH and VL of the antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding the VH of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding the VL of an antibody or antigen-binding fragment thereof described herein. In a specific embodiment, provided herein are polynucleotides 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 the antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein are combinations of two polynucleotides, wherein the first polynucleotide of the combination comprises a first nucleotide sequence encoding the VH of an antibody or antigen-binding fragment thereof described herein, and the second polynucleotide of the combination comprises a second nucleotide sequence encoding the VL of the antibody or antigen-binding fragment thereof. In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding the heavy chain of an antibody described herein. In a specific embodiment, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain of an antibody described herein. In a specific embodiment, a polynucleotide is provided herein, comprising a first nucleotide sequence encoding a heavy chain of an antibody described herein and a second nucleotide sequence encoding a light chain of the antibody. In a specific embodiment, a combination of two polynucleotides is provided herein, wherein the first polynucleotide of the combination comprises a first nucleotide sequence encoding a heavy chain of an antibody described herein, and the second polynucleotide of the combination comprises a second nucleotide sequence encoding a light chain of the antibody.

[0393] In a specific embodiment, the polynucleotide disclosed herein comprises the nucleotide sequence of SEQ ID NO: 23. In a specific embodiment, the polynucleotide disclosed herein comprises the nucleotide sequence of SEQ ID NO: 24. In a specific embodiment, the polynucleotide disclosed herein comprises the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24. In a specific embodiment, the polynucleotide combination disclosed 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.

[0394] Also provided herein are polynucleotides encoding anti-KIT antibodies or fragments thereof that have been optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination 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 eliminating inhibitory regions in mRNA can be performed by adjusting 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 accordingly. For example, potential splice sites and instability elements (e.g., A / T or A / U-rich elements) within the RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of recombinant RNA expression. These changes take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acids. In some embodiments, it may be necessary to change one or more codons to encode a conservative mutation, such as an analogous amino acid having similar chemical structure and properties and / or function as the original amino acid. Such methods can increase expression of the 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 relative to expression of the anti-KIT antibody encoded by a non-optimized polynucleotide.

[0395] In certain embodiments, an optimized polynucleotide sequence encoding an anti-KIT antibody or fragment thereof (e.g., a VL domain and / or a VH domain) described herein can hybridize with an antisense (e.g., complementary) polynucleotide encoding a non-optimized polynucleotide sequence encoding an anti-KIT antibody or fragment thereof (e.g., a VL domain and / or a VH domain) described herein. In a specific embodiment, an optimized nucleotide sequence encoding an anti-KIT antibody or fragment thereof described herein hybridizes under high stringency conditions with an antisense polynucleotide encoding a non-optimized polynucleotide sequence of an anti-KIT antibody or fragment thereof described herein. In a specific embodiment, an optimized nucleotide sequence encoding an anti-KIT antibody or fragment thereof described herein hybridizes under high stringency, medium stringency, or low stringency hybridization conditions with an antisense polynucleotide encoding a non-optimized nucleotide sequence of an anti-KIT antibody or fragment thereof described herein. Information regarding hybridization conditions is described, for example, in U.S. Patent Application Publication No. US2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.

[0396] In certain embodiments, the optimized polynucleotide sequence encoding the VL region of an antibody described herein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the nucleotide sequence of SEQ ID NO: 23. In certain embodiments, the optimized polynucleotide sequence encoding the VH region of an antibody described herein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the nucleotide sequence of SEQ ID NO: 24.

[0397] The polynucleotides can be obtained by any method known in the art and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. The nucleotide sequences encoding the antibodies described herein and modified forms of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in a certain manner to produce nucleic acids encoding the antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., Kutmeier et al., 1994, BioTechniques 17: 242). In short, the assembly involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0398] Alternatively, polynucleotides encoding antibodies described herein can be produced from nucleic acids from suitable sources (e.g., hybridomas) using well-known methods in the art (e.g., PCR and other molecular cloning methods). For example, genomic DNA obtained from hybridoma cells producing antibodies of interest can be used to perform PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of known sequences. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding antibody light chains and / or heavy chains. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding variable light chains and / or variable heavy chains of antibodies. The amplified nucleic acids can be cloned into vectors to be expressed in host cells and further cloned, for example, thereby producing chimeric antibodies and humanized antibodies.

[0399] 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 isolated from a suitable source (e.g., an antibody cDNA library or a cDNA library produced by any tissue or cell expressing the antibody, such as a hybridoma cell selected for expression of the antibodies described herein, or nucleic acid isolated therefrom, preferably polyA+RNA) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, cDNA clones from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0400] DNA encoding the anti-KIT antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the anti-KIT antibodies). Hybridoma cells can be used as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulins, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., from CHO GS System TM (Lonza) CHO cells) or myeloma cells to synthesize anti-KIT antibodies in these recombinant host cells.

[0401] In order to produce complete antibodies, PCR primers comprising VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting restriction sites can be used to amplify the VH or VL sequences in scFv clones. Utilizing 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 (e.g., human γ1 or γ4 constant region), and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region (e.g., human κ or λ constant region). In certain embodiments, the vector for expressing the VH or VL domain comprises an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker (e.g., neomycin). The VH and VL domains can also be cloned into a vector expressing the necessary constant regions. Then, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0402] The DNA also may be modified, for example, by substituting coding sequences for human heavy and light chain constant domains in place of the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0403] 5.3 Host Cells and Recombinant Antibody Expression

[0404] In certain aspects, provided herein are host cells and related expression vectors for recombinantly expressing the antibodies (or antigen-binding fragments thereof) described herein. Provided herein are vectors (e.g., expression vectors) and vector combinations for recombinant expression in host cells, preferably mammalian cells, comprising a polynucleotide comprising a nucleotide sequence encoding an anti-KIT antibody or antigen-binding fragment. Also provided herein are host cells comprising such vectors or vector combinations for recombinantly expressing the anti-KIT antibodies (e.g., human or humanized antibodies) described herein.

[0405] Recombinant expression of antibodies described herein that immunospecifically bind to the KIT antigen (e.g., full-length antibodies, antibody heavy and / or light chains, or single-chain antibodies described herein) involves constructing one or more expression vectors containing polynucleotides encoding the antibodies. After obtaining polynucleotides encoding the antibody molecules, antibody heavy and / or light chains, or fragments thereof (preferably, but not necessarily, containing heavy and / or light chain variable domains) described herein, one or more vectors for producing the antibody molecules can be generated by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies (or VH / VL or heavy / light chains) are described herein. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule, an antibody heavy or light chain, an antibody heavy or light chain variable domain, or fragments thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can, for example, include nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and the variable domains of an antibody can be cloned into such vectors to express the entire heavy chain, the entire light chain, or the entire heavy and light chains.

[0406] In a specific embodiment, a vector is provided herein, comprising a polynucleotide encoding the VH or antigen-binding fragment thereof of an antibody described herein. In a specific embodiment, a vector is provided herein, comprising a polynucleotide encoding the VL or antigen-binding fragment thereof of an antibody described herein. In a specific embodiment, a vector is provided herein, comprising a polynucleotide encoding the VH and VL or antigen-binding fragment thereof of an antibody described herein. In a specific embodiment, a vector is provided herein, comprising a first polynucleotide encoding the VH or antigen-binding fragment thereof of an antibody described herein and a second polynucleotide encoding the VL or antigen-binding fragment thereof of the antibody described herein. In a specific embodiment, a combination of two vectors is provided herein, wherein the first vector in the combination comprises a first polynucleotide encoding the VH or antigen-binding fragment thereof of an antibody described herein, and the second vector in the combination comprises a second polynucleotide encoding the VL or antigen-binding fragment thereof of the antibody described herein. In a specific embodiment, a vector is provided herein, comprising a polynucleotide encoding the heavy chain of an antibody described herein. In a specific embodiment, a vector is provided herein, comprising a polynucleotide encoding the light chain of an antibody described herein. In a specific embodiment, a vector is provided herein, comprising polynucleotides encoding the heavy and light chains of an antibody described herein. In a specific embodiment, provided herein is a vector comprising a first polynucleotide encoding the VH of an 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, wherein the first vector in the combination comprises a first polynucleotide encoding the VH of an antibody described herein, and the second vector in the combination comprises a second polynucleotide encoding the VL of the antibody.

[0407] The expression vector or the combination of expression vectors can be transferred into a cell (such as a host cell) by conventional techniques, and then the resulting cells can be cultured by conventional techniques to produce an antibody described herein or its fragment. Therefore, there is provided herein a host cell, the host cell containing polynucleotides or polynucleotide combinations encoding an antibody described herein or its fragment, or its heavy chain or light chain, or its fragment, or a single-chain antibody described herein, the polynucleotides or polynucleotide combinations being operably connected to a promoter to express these sequences in the host cell. In certain embodiments, in order to express a double-chain antibody, the vectors encoding the heavy chain and light chain respectively can be co-expressed in the host cell to express the entire immunoglobulin molecule, as will be described in detail below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding the heavy chain and light chain (or VH and VL) of an antibody described herein or its fragment. In a specific embodiment, the host cell contains two different vectors, a first vector comprising a polynucleotide encoding the heavy chain (or VH) of an antibody described herein or its fragment, and a second vector comprising a polynucleotide encoding the light chain (or VL) of an antibody described herein or its fragment. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain (or VH) or a 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 a fragment thereof of an antibody described herein.

[0408] 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). These host-expression systems represent vehicles that can produce and subsequently purify the coding sequence of interest, and also represent cells that can in situ express the antibody molecules described herein when transformed or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms, such as bacteria (e.g., Escherichia coli and Bacillus subtilis (B. subtilis)) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Pichia pastoris (Saccharomyces Pichia)) transformed with recombinant yeast expression vectors containing the antibody coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequence; virus (CaMV); tobacco mosaic virus (TMV)) or plant cell systems (e.g., green algae, such as Chlamydomonas reinhardtii) transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3 cells) with 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., adenovirus late promoter; vaccinia virus 7.5K promoter). In a specific embodiment, the cells used to express the antibodies or antigen-binding fragments thereof described herein are CHO cells, such as those from the CHO GS System. TM In one embodiment, the mammalian expression vector is pOptiVEC TMOr pcDNA3.3. Preferably, bacterial cells (e.g., E. coli) are used, and more preferably, eukaryotic cells (particularly for expressing whole recombinant antibody molecules) are used to express the recombinant antibody molecules. For example, mammalian cells (e.g., Chinese hamster ovary (CHO) cells) in combination with vectors (e.g., major immediate early gene promoter elements from human cytomegalovirus) are effective antibody expression systems (Foecking et al., 1986, Gene 45: 101; and Cockett et al., 1990, Bio / Technology 8: 2). In certain 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 antibody described herein that immunospecifically binds to the KIT antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0409] In bacterial systems, a variety of expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when large quantities of such antibodies are to be produced, vectors that direct high-level expression of readily purified fusion protein products may be desirable in order to produce pharmaceutical compositions of the antibody molecule. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)), in which the antibody coding sequence can be ligated separately into the vector in frame with the lacZ coding region to produce a fusion protein; and pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509). pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites, thereby allowing the cloned target gene product to be released from the GST moiety.

[0410] In insect systems, the Autographaca ifornica nuclear polyhedrosis virus (AcNPV) has been used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. Antibody coding sequences can be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0411] In mammalian host cells, many viral-based expression systems can be utilized. When using adenovirus as an expression vector, the antibody coding sequence of interest can be connected to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. This chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will produce a recombinant virus that is viable and capable of expressing antibody molecules in infected hosts (e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 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. In addition, the initiation codon must be synchronized with the reading frame of the desired coding sequence to ensure translation of the entire inserted sequence. These exogenous translation control signals and initiation codons can have a variety of sources, such as natural and synthetic sources. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153: 51-544).

[0412] In addition, the expression of the inserted sequence can be selected or the host cell strain of modifying and processing the gene product in a required ad hoc manner can be selected. These modifications (such as glycosylation) and processing (such as cracking) for the protein product may be extremely important for the function of the protein. Different host cells have unique specific mechanisms for the post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein. For this reason, the eukaryotic host cell used can have the cellular machinery for the appropriate processing of the primary transcript, the glycosylation of the gene product and the phosphorylation. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (mouse myeloma cell line that does not endogenously produce any immunoglobulin chain), CRL7030 and HsS78Bst cells. In certain embodiments, the humanized monoclonal anti-KIT antibodies described herein are produced in mammalian cells (e.g., CHO cells).

[0413] In order to produce recombinant protein for a long time and in high yield, preferably stably expressed. For example, the cell line of stably expressed antibody molecules can be engineered. Host cells can be transformed with DNA and selective markers controlled by appropriate expression control elements (such as promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), rather than using expression vectors containing viral replication origins. After introducing foreign DNA, the engineered cells can be grown for 1-2 days in enriched medium (enriched media) and then transferred to selective medium. The selective marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosome and grow to form plaques, which can be cloned and expanded into cell lines. This method can be advantageously used to engineer the cell line expressing antibody molecules. This type of engineered cell line is particularly suitable for screening and evaluating compositions that directly or indirectly interact with antibody molecules.

[0414] A variety of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17). The genes can be used for tk- cells, hgprt- cells, or aprt- cells, respectively. In addition, antimetabolite resistance can also be used as the basis for selection for 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); nco, 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). Methods generally known in the art of recombinant DNA technology can be routinely used to select the desired recombinant clones and are 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 Dracopoli et al. (eds.), Current Protocols in Human Genetics , John Wiley & Sons, NY (1994), Chapters 12 and 13; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, which are incorporated herein by reference in their entirety.

[0415] The expression level of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel. The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker in the vector system for expressing the antibody is amplifiable, an increase in the level of inhibitors 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, the production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

[0416] Host cells can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors can contain the same selectable marker, thereby enabling equal expression of the heavy and light chain polypeptides. Host cells can be co-transfected with different amounts of the two or more expression vectors. For example, the host cells can be transfected with the first expression vector and the second expression vector in any of the following ratios: 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.

[0417] Alternatively, a single vector can be used that encodes and is capable of expressing both heavy and light chain polypeptides. In this case, the light chain should be placed before the heavy chain to avoid excess non-toxic 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. The polycistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can comprise a promoter, a first gene (e.g., a heavy chain of an antibody described herein), and a second gene (e.g., a light chain of an antibody described herein) in the following order. In such an expression vector, transcription of both genes can be driven by the promoter, while translation of the mRNA of the first gene can occur by a cap-dependent scanning mechanism, and translation of the mRNA of the second gene can occur by a cap-independent mechanism, such as through an IRES.

[0418] After the antibody molecules described herein have been produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange chromatography; affinity chromatography, particularly for specific antigens after protein A; and size-restricted column chromatography), centrifugation, differential solubility methods, or by any other standard technique for protein purification. In addition, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0419] In a specific embodiment, the antibodies described herein are separated or purified. In general, an isolated antibody is an antibody that is substantially free of other antibodies having an antigenic specificity different from that of the isolated antibody. For example, in a specific embodiment, the preparation of the antibodies described herein is substantially free of cellular material and / or chemical precursors. The phrase "substantially free of cellular material" includes antibody preparations in which the antibody is separated from or recombinantly produces the cellular components of the cells of the antibody. Therefore, antibodies that are substantially free of cellular material include antibody preparations with 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 antibody variants, such as different post-translational modified forms of antibodies or other different forms of antibodies (e.g., antibody fragments). When the antibody is produced recombinantly, it is generally also substantially free of culture medium, that is, culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5% or 0.1% of the protein preparation volume. When an 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 protein synthesis. Thus, such antibody preparations contain 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 antibodies described herein are isolated or purified.

[0420] 5.4 Antibody Production

[0421] Antibodies (e.g., human or humanized antibodies) (or antigen-binding fragments thereof) that immunospecifically bind to a KIT antigen as described herein can be produced by any antibody synthesis method known in the art, such as by chemical synthesis or by recombinant expression techniques. In a specific aspect, provided herein are methods for preparing the antibodies described herein, comprising culturing and / or expressing such antibodies using host cells as described herein, the methods optionally further comprising purifying the antibodies obtained from the host cells. Unless otherwise indicated, the methods described herein employ conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of 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 annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (ed.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor, NY. Spring Harbor Laboratory Press.

[0422] For example, humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089); veneering or resurfacing (European Patent Nos. 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 91:969-973); chain shuffling (U.S. Patent No. 5,565,332); and, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, WO 9317105; Tan et al., J. Immunol. 169: 111925 (2002); Caldas et al., Protein Eng. 13(5): 353-60 (2000); Morea et al., Methods 20(3): 26779 (2000); Baca et al., J. Biol. Chem. 272(16): 10678-84 (1997); Roguska et al., Protein Eng. 9(10): 895904 (1996); Couto et al., Cancer Res. 55(23 Suppl): 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 No. US2005 / 0042664A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0423] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma technology, recombinant technology and phage display technology, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including technology known and taught in the art, such as Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563681 (Elsevier, NY, 1981). As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced by recombinant technology, such as recombinant monoclonal antibodies expressed by host cells (e.g., mammalian host cells).

[0424] 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 appropriate host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., the extracellular domain of human KIT). Alternatively, lymphocytes can 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)). In addition, animals can also be immunized using the repeated immunization with multiple sites (RIMMS) technique (Kilptrack et al., 1997 Hybridoma 16: 381-9, which is incorporated herein by reference).

[0425] Non-limiting examples of myeloma cell lines include murine myeloma lines, such as the MOPC-21 and MPC-11 mouse tumor cell lines obtained from the Salk Institute Cell Distribution Center in San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells obtained from the American Type Culture Collection in 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)).

[0426] The antibodies described herein include antibody fragments that recognize specific KIT antigens and can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (for producing Fab fragments) or pepsin (for producing F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The AF(ab')2 fragment contains the two antigen-binding arms of the antibody molecule connected by a disulfide bond in the hinge region.

[0427] In one aspect, to generate complete antibodies, PCR primers comprising VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify the VH or VL sequence from a template, such as an scFv clone. Utilizing 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 a 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 using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0428] 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): 277-302; U.S. Pat. No. 6,005,079; and International Publication Nos. WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301.

[0429] 5.5 Treatment methods and medical uses

[0430] Provided herein are methods for inhibiting, preventing, prophylactic, treating, and / or managing KIT-related conditions or diseases. Such methods comprise 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 aspects, provided herein are methods for inhibiting, preventing, prophylactic, treating, or managing one or more symptoms of a KIT-related condition or disease.

[0431] In specific embodiments, the methods described herein for treating a KIT-related condition or disease provide for a reduction or improvement in the progression, severity, and / or duration of a KIT-related condition or disease by administering one or more therapies, including but not limited to administration of one or more prophylactic or therapeutic agents, such as the anti-KIT antibodies described herein or the pharmaceutical compositions described herein. In other specific embodiments, the methods described herein for treating a KIT-related condition or disease involve alleviating one or more symptoms of a KIT-related condition or disease. In specific embodiments, an antibody or antigen-binding fragment thereof, or conjugate thereof, or a pharmaceutical composition described herein is used to prevent, treat, or manage a KIT-related condition. In a specific embodiment, the KIT-related disease or condition treated, managed, or prevented with an anti-KIT antibody or antigen-binding fragment thereof, or conjugate thereof, or a pharmaceutical composition described herein is related to KIT expression and / or activity, e.g., involves cells expressing KIT and / or exhibiting KIT activity, but is not caused or resulting from KIT expression or activity.

[0432] In a specific embodiment, the antibody used in the methods described herein is internalized by the cell to which the antibody binds. In a specific 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 (e.g., a toxin). In a certain embodiment, the conjugate used in the methods described herein is internalized into the cell to which the conjugate binds.

[0433] In certain embodiments, KIT is abnormally (e.g., highly) expressed by a cell, 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 a control cell (e.g., a cell expressing normal levels of KIT, e.g., a normal, e.g., human, mast cell, stem cell, brain cell, melanoblast, or ovarian cell). In certain embodiments, KIT expression results in cell surface KIT expression that is 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, stem cell, brain cell, melanoblast, or ovarian cell population). In specific embodiments, such control cells can be obtained or derived from a healthy individual (e.g., a healthy human). In some embodiments, KIT may 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 may be abnormally upregulated in a particular cell type, regardless of whether KIT is abnormally expressed on the cell surface. In certain embodiments, KIT signaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than KIT signaling in a control cell (e.g., a cell containing normal KIT signaling, such as a mast cell, stem cell, brain cell, melanoblast, or ovarian cell). In certain embodiments, KIT signaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the average KIT signaling in a control cell population (e.g., a cell population exhibiting normal KIT signaling, such as a normal, e.g., human, mast cell population, stem cell population, brain cell population, melanoblast population, or ovarian cell population). In certain embodiments, normal, abnormal, or excessive cell signaling is caused by KIT binding to a KIT ligand. In other embodiments, abnormal or excessive cell signaling occurs independent of KIT binding to a KIT ligand.

[0434] In certain aspects, a KIT-related condition or disease can be characterized by gain-of-function KIT activity, increased KIT activity, or overexpression of KIT. In one embodiment, a KIT-related condition or disease is caused in whole or in part by, or is the result of, gain-of-function KIT activity or expression (e.g., overexpression of KIT). In certain embodiments, gain-of-function KIT activity may occur independently of binding of a KIT ligand (e.g., SCF) to a KIT receptor. In certain aspects, high expression or overexpression of KIT in a cell refers to an expression level that is higher than the expression level of a reference cell known to have normal KIT expression or KIT activity, or that is at least about 35%, 45%, 55%, or 65% higher than the average expression level of KIT in a cell population or sample 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, a KIT-related condition or disease is characterized by KIT activity that is higher than normal KIT activity and contributes to cellular transformation, neoplasia, and tumorigenesis. In certain aspects, high or increased KIT activity in a cell refers to a level of KIT activity that is at least about 35%, 45%, 55%, or 65% higher than the expression level in a reference cell known to have normal KIT activity, or higher than the average level of KIT activity in a population of cells or a sample known to have normal KIT activity. Non-limiting examples of KIT activity include tyrosine phosphorylation of the cytoplasmic domain of KIT, and signaling downstream of KIT, such as Stat or Akt signaling.

[0435] In certain embodiments, the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In certain embodiments, the KIT-associated disorder is a fibrotic or inflammatory disease, such as inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC). In other embodiments, the KIT-associated disease is a cancer, such as lung cancer (e.g., small cell lung cancer), leukemia, neuroblastoma, melanoma, sarcoma (e.g., Ewing's sarcoma), or gastrointestinal stromal tumor (GIST). In other embodiments, the KIT-associated disease is a systemic mast cell disease (e.g., mastocytosis), a blood disorder, fibrosis (e.g., idiopathic pulmonary fibrosis (TPF), scleroderma, or myelofibrosis), or an inflammatory disorder, such as asthma, rheumatoid arthritis, inflammatory bowel disease, or allergic inflammation.

[0436] 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).

[0437] Mast cells, derived from bone marrow progenitors, are large cells found in connective tissues throughout the body, with the highest abundance in the submucosal tissues and dermis. They contain large granules that store a variety of mediator molecules, including the vasoactive amine histamine, and possess high-affinity Fes receptors (FcsRI), enabling them to bind IgE monomers. Antigen binding to mast cell-bound IgE triggers mast cell degranulation and activation, resulting in localized or systemic immediate hypersensitivity reactions. Therefore, mast cells play a crucial role in inflammatory and allergic responses. However, without proper balance and regulation, mast cells can also cause harmful overreactions to antigens observed in conditions such as allergy, atopy, and rhinitis.

[0438] KIT signaling is important for mast cell development and homeostasis, such as the expansion of mast cells from their progenitors and their subsequent maturation and survival in their resident tissues, mast cell homing to their resident sites in the body, and promoting mast cell adhesion to extracellular matrix proteins. Activating mutations of KIT, such as those at amino acid residues 816 or 560 in KIT, are associated with mastocytosis and gastrointestinal stromal cell tumors (GISTs), which are characterized by overproduction of mast cells.

[0439] In a specific embodiment, the methods described herein for preventing, treating, or managing a KIT-mediated disorder, such as a fibrotic or inflammatory disorder (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation) in a subject in need thereof can achieve at least one, two, three, four, or more of the following effects by administering a therapeutically effective amount of an anti-KIT antibody described herein or a pharmaceutical composition described herein: (i) reducing or ameliorating the severity of the fibrotic or inflammatory disorder (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 the fibrotic or inflammatory disorder (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (iii) preventing the recurrence of the fibrotic or inflammatory disorder (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (iv) reducing the subject's hospitalizations; (v) reducing the length of hospitalization; (vi) inhibiting (e.g., partially inhibiting) the onset of the disease; (x) reducing the rate of hospitalizations; (xi) reducing the number of symptoms associated with a fibrotic or inflammatory disorder (e.g., asthma, 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 suffering from a fibrotic or inflammatory disorder (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).

[0440] Other non-limiting examples of KIT-associated disorders or diseases include systemic mast cell disorders (e.g., mastocytosis), hematological disorders, fibrosis (e.g., idiopathic pulmonary fibrosis (TPF), scleroderma, or myelofibrosis), and inflammatory conditions, such as asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation.

[0441] As used herein, the term "mast cell related disorder" or "mast cell related disorders" refers to a condition in which mast cell activity leads to pathological changes and / or abnormal amounts (e.g., higher than normal or lower than normal) of mast cells are found in various parts of the body. For example, a mast cell related disorder can manifest as the accumulation of pathological mast cells in virtually any or all organs and tissues and / or the abnormal release of one or more mast cell mediators (e.g., inflammatory mediators). Non-limiting examples of inflammatory mediators released by mast cells include any of the following: (i) granule-associated mediators, including histamine, serotonin (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 alpha (TNFa), and chemokines, including CCL-2, CCL-3, CCL-5, and CXCL8.

[0442] In a specific aspect, the mast cell-associated disorder is a mast cell-associated disorder of the nervous system (eg, central nervous system) such as NMO, NMOSD, MS, or NF (eg, NF type 1 (NF1), NF type 2 (NF2), or schwannoma).

[0443] MS is a chronic inflammatory demyelinating disease of the central nervous system (brain and spinal cord) in which the white matter in the patient's brain or spinal cord becomes inflamed and then damaged by the individual's own immune system. These inflamed areas leave scars in the brain and spinal cord. This damage disrupts the ability of the various parts of the nervous system to communicate, leading to a variety of symptoms, including physical, psychological and / or psychiatric problems. Forms of MS include, but are not limited to, relapsing (symptoms occur in isolated attacks) and progressive (symptoms worsen over time). Guidelines for diagnosing MS have been described, see, for example, 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 Guidelines, No. 8.), available at: http: / / www.ncbi.nlm.nih.gov / books / NBK48919 / . Symptoms of MS can manifest as any neurological symptom or sign, such as autonomic, visual, motor, and sensory problems. Non-limiting examples of MS symptoms include loss of sensitivity or changes in sensation (e.g., tingling, pins and needles, or numbness), muscle weakness, very pronounced reflexes, muscle spasms or difficulty moving, coordination and balance difficulties (ataxia), speech or swallowing problems, visual problems (nystagmus, optic neuritis, or diplopia), fatigue, acute or chronic pain, bladder and bowel difficulties, mood problems (e.g., depression or mood swings), Uhthoff phenomenon (worsening of symptoms due to exposure to higher than usual temperatures), and Lhermitte's sign (a sensation of an electric shock conducted down the back when bending the neck). In particular aspects, provided herein are methods of preventing, treating, alleviating, or managing one or more of these MS symptoms 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).

[0444] Neuromyelitis optica (NMO), or Devic's disease, is an autoimmune inflammatory disorder of the central nervous system that primarily affects the optic nerves and spinal cord, and in some cases, the brain. NMO can cause paralysis and blindness. Most patients with NMO are seropositive for immunoglobulin autoantibodies against aquaporin-4 (AQP4), a water channel widely expressed in the optic nerves, spinal cord, and periventricular regions (AQP4-IgG or NMO-IgG). A small percentage of patients with NMO are NMO-IgG negative.

[0445] NMOSD refers to a variety of conditions that are associated with NMO but may not fully meet the clinical diagnostic criteria for a definitive diagnosis of NMO. Non-limiting examples of conditions that are typically included in this NMOSD category include NMO-IgG seropositive restricted NMO (e.g., single or recurrent longitudinally extending long segment transverse myelitis (LETM) [e.g., spinal cord lesions of ≥3 vertebral segments visible on MRI), recurrent or simultaneous bilateral optic neuritis (ON)], Asian optometrospinal MS (OSMS), optic neuritis or LETM associated with systemic autoimmune disease, and optic neuritis or myelitis associated with typical brain lesions of NMO (e.g., hypothalamic or brainstem lesions) (see, e.g., Oh et al., Neurology Research International, Vol. 2012, Article ID 460825, p. 13, 2012).

[0446] In certain aspects, diagnostic criteria for NMO include, but are not limited to, the presence of myelitis and optic neuritis, and any two of the following: (i) extended myelitis visible on spinal cord MRI; (ii) normal brain MRI at onset; and (iii) positive anti-AQP4 antibodies (see, e.g., Collongues et al., Ther. Adv. Neurol. Disotd., 2011, 4: 111-121).

[0447] Non-limiting examples of NMO or NMOSD symptoms include acute optic neuritis (e.g., bilateral), transverse myelitis (e.g., longitudinal extension), unilateral or bilateral visual loss, eye pain, severe paraplegia, asymmetric sensory levels, bladder dysfunction, paroxysmal tonic spasms of the trunk and limbs, and Lhermitte phenomenon. In some aspects, cervical spinal cord lesions extending rostral to the cervicomedullary junction can cause symptoms such as acute respiratory decompensation, nausea, intractable vomiting, and hiccups. In some aspects, hypothalamic-pituitary axis dysfunction associated with NMO can manifest as lethargy, hyponatremia, hypothermia, hypothyroidism, and hyperprolactinemia. In addition, confusion, sudden changes in level of consciousness, cortical blindness, and imaging findings suggestive of posterior reversible encephalopathy syndrome (PRES) may also be associated with NMO.

[0448] NF is a genetic disorder of the nervous system that primarily affects the development and growth of nervous system (nerve) tissue and causes tumors called neurofibromas to grow along the body's nerves. Although NF is generally a hereditary disorder, new cases can arise spontaneously through genetic mutations. NF is usually diagnosed in childhood, between the ages of 3 and 16, and can sometimes be diagnosed in infancy (for children with severe disease).

[0449] Non-limiting types of NF include NF type 1 (NF1), NF type 2 (NF2), and schwannomatosis. NF1 is also known as von Recklinghausen disease. The phenotypic manifestations of NF1 include light brown skin spots that appear at birth or in childhood, neurofibromas (tumors that grow along the subcutaneous nerves, also known as dermal neurofibromas), plexiform neurofibromas (tumors that involve multiple nerves), spinal cord and optic nerve tumors, and learning disabilities. In some aspects, individuals affected by NF1 have a greater probability of developing gastrointestinal stromal tumors (GISTs) than the general population. Neurofibromas can be considered to be external neurofibromas, such as cutaneous or dermal neurofibromas, or can be considered to be internal neurofibromas, such as plexiform neurofibromas.

[0450] NF2 is an autosomal dominant inherited disorder associated with neurological, ophthalmic, and skin abnormalities. Non-limiting examples of NF2 symptoms include hearing loss, tinnitus, visual impairment, imbalance, and painful skin lesions. In certain aspects, skull base tumors (including vestibular schwannomas (VS) and meningiomas) in NF2 patients can lead to lower cranial nerve dysfunction and death.

[0451] The diagnosis of NF2 can be established by the presence of bilateral vestibular schwannomas (VS) or unilateral VS and a family history of NF2-related tumors (such as meningiomas, schwannomas, ependymomas, gliomas, or neurofibromas), posterior polar cataracts, or other NF2-related tumors. In addition to the morbidity associated with auditory and vestibular dysfunction, patients may also experience other neurological deficits related to the growth of the VS (such as those due to compression of other cranial nerves).

[0452] Schwannomatosis shares many features with the more well-known form of NF. Multiple schwannomas or nerve sheath tumors are seen in schwannomatosis, but the characteristic vestibular (otitis nerve) tumors seen in NF2 are not present. In certain aspects, patients with schwannomatosis develop tumors on the nerve sheaths or coverings (see, e.g., MacCollin et al., Neurology, 2005, 64:1838-1845).

[0453] Other non-limiting examples of mast cell-associated disorders include, for example, anaphylaxis, atopic diseases, mast cell activation syndrome, allergic rhinitis, food and venom-related allergies (e.g., allergies to tree nuts, shellfish, fish, hymenopteran venom, or bee stings), 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., due to 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, such as angioedema and anaphylaxis.

[0454] Alternatively, mast cell related conditions can be urticaria, particularly chronic urticaria, including chronic spontaneous urticaria (CSU), chronic idiopathic urticaria and chronic induced urticaria (chronic induced urticaria) (i.e., chronic inducible urticaria (CIndU)). In a specific embodiment, mast cell related conditions are chronic spontaneous urticaria. In a specific embodiment, mast cell related conditions are moderate to severe chronic spontaneous urticaria. Chronic spontaneous urticaria is characterized by the occurrence of wheals or urticaria, which last for 6 weeks or longer, without identifiable specific inducements or causes. In a specific embodiment, mast cell related conditions are chronic induced urticaria. Chronic induced urticaria is a form of urticaria with an attributable triggering factor, which typically results in wheals (wheals) or angioedema. In a specific embodiment, chronic induced urticaria is cold urticaria (ColdU). People with cold urticaria experience symptoms such as itching, burning wheals, and angioedema when their skin is exposed to temperatures below skin temperature. In another embodiment, chronic induced urticaria is symptomatic dermatographism (SD). Symptomatic dermatographism is characterized by wheals and erythema reactions that occur when the skin is gently stroked, scratched, or rubbed, and usually occurs within a few minutes of stimulation. In another embodiment, chronic induced urticaria is cholinergic urticaria. Cholinergic urticaria is triggered by the body's sweating response to active or passive body warming and is characterized by small (1-4 mm) wheals surrounded by bright red erythema. Common triggers include exercise, hot baths / showers, fever, occlusive dressings, eating spicy foods, and emotional stress. In another embodiment, chronic induced urticaria is heat urticaria. In another embodiment, chronic induced urticaria is delayed pressure urticaria. In another embodiment, chronic induced urticaria is solar urticaria. In another embodiment, chronic induced urticaria is vibratory urticaria. In another embodiment, chronic induced urticaria is contact urticaria. In another embodiment, chronic induced urticaria is aquagenic urticaria. Antihistamines are approved therapies for chronic induced urticaria.

[0455] Mast cell-related disorders may also be chronic prurigo. In specific embodiments, the chronic prurigo is prurigo nodularis. In specific embodiments, the chronic prurigo is prurigo epidemicis. In specific embodiments, the chronic prurigo is prurigo nodularis. In specific embodiments, the chronic prurigo is prurigo plaques. In specific embodiments, the chronic prurigo is prurigo umbilicus. In specific embodiments, the chronic prurigo is prurigo linearis. In some embodiments, the patient exhibits a monotypic lesion phenotype. In other embodiments, the patient exhibits a polytypic lesion phenotype.

[0456] In various embodiments, the patient experience of mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) for one or more previous treatments of this illness has failed. In certain embodiments, one or more previous treatments include at least one standard care therapy for this illness. In certain embodiments, one or more previous treatments are all standard care therapy for this illness. In certain embodiments, the patient experience of mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) has failed to treat this illness with antihistamines. In specific embodiments, the patient experience of mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) has failed to treat this illness with H1-antihistamines. In a specific embodiment, the patient suffering from a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed to treat the disorder with an H2-antihistamine. In a specific embodiment, the patient suffering from a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed to treat the disorder with both H1- and H2-antihistamines. In certain embodiments, the patient suffering from a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed to treat the disorder with one or more leukotriene receptor antagonists. In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with one or more immunomodulatory agents or anti-inflammatory agents. In specific embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab. In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab. In specific embodiments, a patient with a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed treatment for the disorder with an IL-5R inhibitor, such as an anti-IL-5R antibody, such as benralizumab. In a specific embodiment, a patient with a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed treatment for the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, such as mepolizumab. In a specific embodiment, a patient with a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed treatment for the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, such as lirentelimab. In certain embodiments, a patient with a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) has failed treatment for the disorder with a TSLP or TSLPR inhibitor, such as an anti-TSLP antibody or an anti-TSLPR antibody, such as tezepelumab (Tezspire TMIn a specific embodiment, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) and has failed treatment for the disorder with a C5aR inhibitor, such as an anti-C5aR antibody, such as avdoralimab. In a specific embodiment, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) and has failed treatment for the disorder with a CD200R inhibitor, such as an anti-CD200R antibody, such as LY3454738. In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with one or more Bruton's Tyrosine Kinase (BTK) inhibitors, such as remibrutinib and / or rilzabrutinib. In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); (2) treatment with one or more leukotriene receptor antagonists; (3) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, such as anti-IgE antibodies, such as omalizumab); or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinib, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738); and / or (4) treatment with one or more BTK inhibitors (e.g., ribrutinib and / or rizabrutinib). In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); and (2) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, e.g., anti-IgE antibodies, e.g., omalizumab). or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM ); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinimab, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738). In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); (2) treatment with an IgE inhibitor, e.g., an anti-IgE antibody, e.g., omalizumab or ligralizumab; and (3) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab. In certain embodiments, the patient has a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) who has failed treatment for the disorder with: (1) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab or ligralizumab; and (2) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder is chronic prurigo, and the patient is treated with an IL-4R inhibitor (e.g., an anti-IL-4R antibody, e.g., dupilumab). ) treatment has failed. In certain embodiments, the mast cell-related disorder is chronic prurigo, and the patient has failed treatment with an IL-31 receptor alpha inhibitor (e.g., an anti-IL-31 receptor alpha antibody, such as nemolizumab). In certain embodiments, the mast cell-related disorder is chronic prurigo, and the patient has failed treatment with a Janus kinase 1 inhibitor (e.g., INCB054707 or abrocitinib). In certain embodiments, the mast cell-related disorder is chronic prurigo, and the patient has failed treatment with a neurokinin-1 (NK1) receptor antagonist (e.g., serlopitant). In certain embodiments, the mast cell-related disorder is chronic prurigo, and the patient has failed treatment with a PDE4 and / or TNF-alpha inhibitor (e.g., apremilast). In certain embodiments, the mast cell-related disorder is chronic prurigo, and the patient has failed treatment with an OSMRβ inhibitor (e.g., an anti-OSMRβ antibody, such as vixarelimab). In certain embodiments, the mast cell-related disorder is chronic prurigo, and the patient has failed one, two, three or more of the above-mentioned treatments for chronic prurigo.

[0457] A patient is considered to have failed treatment if their condition is refractory to treatment with a treatment directed at the condition, is resistant to treatment, relapses after treatment, and / or if the patient discontinues treatment due to intolerance of treatment.

[0458] In various embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are difficult to treat for one or more previous treatments of this disease. In certain embodiments, one or more previous treatments include at least one standard care therapy for this disease. In certain embodiments, one or more previous treatments are all standard care therapy for this disease. In certain embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are difficult to treat with antihistamines. In specific embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are difficult to treat with H1-antihistamines. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with H2-antihistamines. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with H1- and H2-antihistamines. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with one or more leukotriene receptor antagonists. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with one or more immunomodulatory agents or anti-inflammatory agents. In specific embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab. In certain embodiments, a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) is treated with an IL-5R inhibitor, such as an anti-IL-5R antibody, such as lumefantrine. refractory to treatment. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, such as mepolizumab. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with a Siglec 8 inhibitor, such as an anti-Siglec8 antibody, such as larelizumab. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with a TSLP or TSLPR inhibitor, such as an anti-TSLP antibody or an anti-TSLPR antibody, such as tezelumab (Tezspire TM ) treatment is refractory to treatment with a C5aR inhibitor, such as an anti-C5aR antibody, such as idolinib. In a specific embodiment, the mast cell-related disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-related disorder (e.g., eosinophilic esophagitis) is refractory to treatment with a CD200R inhibitor, such as an anti-CD200R antibody, such as LY3454738. In certain embodiments, the mast cell-related disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-related disorder (e.g., eosinophilic esophagitis) is refractory to treatment with one or more BTK inhibitors, such as ribrutinib and / or rizabrutinib. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); (2) treatment with one or more leukotriene receptor antagonists; (3) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, such as anti-IgE antibodies, such as omalizumab); or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM ); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinib, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738); and / or (4) treatment with one or more BTK inhibitors (e.g., ribrutinib and / or rizabrutinib). In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); and (2) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, e.g., anti-IgE antibodies, e.g., omalizumab). or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM ); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinimab, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); (2) treatment with an IgE inhibitor, e.g., an anti-IgE antibody, e.g., omalizumab or ligralizumab; and (3) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with: (1) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab or ligralizumab; and (2) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder is chronic prurigo and is treated with an IL-4R inhibitor (e.g., an anti-IL-4R antibody, e.g., dupilumab). ) treatment. In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory to treatment with an IL-31 receptor alpha inhibitor (e.g., an anti-IL-31 receptor alpha antibody, such as nemolizumab). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory to treatment with a Janus kinase 1 inhibitor (e.g., INCB054707 or abrocitinib). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory to treatment with a neurokinin-1 (NK1) receptor antagonist (e.g., serlopitant). In certain embodiments, chronic prurigo is refractory to treatment with a PDE4 and / or TNF-α inhibitor (e.g., apremilast). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory to treatment with an OSMRβ inhibitor (e.g., an anti-OSMRβ antibody, such as vesalizumab). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory to treatment with one, two, three, or more of the above-mentioned treatments for chronic prurigo.

[0459] In various embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are resistant to one or more previous treatments for this disease. In certain embodiments, one or more previous treatments include at least one standard care therapy for this disease. In certain embodiments, one or more previous treatments are all standard care therapy for this disease. In certain embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are resistant to antihistamine treatment. In specific embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are resistant to H1-antihistamine treatment. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with an H2-antihistamine. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with both H1- and H2-antihistamines. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with one or more leukotriene receptor antagonists. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with one or more immunomodulatory agents or anti-inflammatory agents. In specific embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with an IL-5R inhibitor, such as an anti-IL-5R antibody, such as lumefantrine. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, such as mepolizumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, such as lerilizumab. In specific embodiments, a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) is treated with a TSLP or TSLPR inhibitor, such as an anti-TSLP antibody or an anti-TSLPR antibody, such as tezemab (Tezspirium). TM ) treatment. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with a C5aR inhibitor, such as an anti-C5aR antibody, such as idolinib. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with a CD200R inhibitor, such as an anti-CD200R antibody, such as LY3454738. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with one or more BTK inhibitors, such as ribrutinib and / or rizabrutinib. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); (2) treatment with one or more leukotriene receptor antagonists; (3) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, such as anti-IgE antibodies, such as omalizumab); or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM ); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinib, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738); and / or (4) treatment with one or more BTK inhibitors (e.g., ribrutinib and / or rizabrutinib). In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with: (1) antihistamine treatment (e.g., H1- and / or H2-antihistamine treatment); and (2) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, e.g., anti-IgE antibodies, e.g., omalizumab). or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, (e.g., mepolizumab; Siglec 8 inhibitors, (e.g., anti-Siglec 8 antibodies, such as lerelizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM ); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinimab, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); (2) treatment with an IgE inhibitor, e.g., an anti-IgE antibody, e.g., omalizumab or ligralizumab; and (3) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is resistant to treatment with: (1) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab or ligralizumab; and (2) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder is chronic prurigo and is responsive to treatment with an IL-4R inhibitor (e.g., an anti-IL-4R antibody, e.g., dupilumab). ) treatment. In certain embodiments, the mast cell-related disorder is chronic prurigo and is resistant to treatment with an IL-31 receptor alpha inhibitor (e.g., an anti-IL-31 receptor alpha antibody, such as nemolizumab). In certain embodiments, the mast cell-related disorder is chronic prurigo and is resistant to treatment with a Janus kinase 1 inhibitor (e.g., INCB054707 or abrocitinib). In certain embodiments, the mast cell-related disorder is chronic prurigo and is resistant to treatment with a neurokinin-1 (NK1) receptor antagonist (e.g., serlopitant). In certain embodiments, the mast cell-related disorder is chronic prurigo and is resistant to treatment with a PDE4 and / or TNF-alpha inhibitor (e.g., apremilast). In certain embodiments, the mast cell-related disorder is chronic prurigo and is resistant to treatment with an OSMRβ inhibitor (e.g., an anti-OSMRβ antibody, such as vesalizumab). In certain embodiments, the mast cell-associated disorder is prurigo chronica and is resistant to treatment with one, two, three or more of the above-mentioned treatments for prurigo chronica.

[0460] In various embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are difficult to treat and have resistance to one or more previous treatments for this disease.In certain embodiments, one or more previous treatments include at least one standard care therapy for this disease.In certain embodiments, one or more previous treatments are all standard care therapy for this disease.In certain embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are difficult to treat and have resistance to antihistamine treatment.In certain embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are difficult to treat and have resistance to H1-antihistamine treatment. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with H2-antihistamines. In specific embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with H1- and H2-antihistamines. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with one or more leukotriene receptor antagonists. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory and resistant to treatment with one or more immunomodulatory agents or anti-inflammatory agents. In specific embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is treated with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is treated with an IL-5R inhibitor, such as an anti-IL-5R antibody, such as lumefantrine. refractory to and resistant to treatment. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, such as mepolizumab. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, such as lareximumab. In specific embodiments, a mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) is treated with a TSLP or TSLPR inhibitor, such as an anti-TSLP antibody or an anti-TSLPR antibody, such as tezemab (Tezspirium). TM) treatment is refractory and resistant to treatment with a C5aR inhibitor, such as an anti-C5aR antibody, such as idolinib. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory and resistant to treatment with a C5aR inhibitor, such as an anti-C5aR antibody, such as idolinib. In a specific embodiment, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory and resistant to treatment with a CD200R inhibitor, such as an anti-CD200R antibody, such as LY3454738. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with one or more BTK inhibitors, such as ribrutinib and / or rizabrutinib. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to treatment with: (1) antihistamine treatment (e.g., H1- and / or H2-antihistamine treatment); (2) treatment with one or more leukotriene receptor antagonists; (3) treatment with one or more immunomodulators or anti-inflammatory agents (e.g., IgE inhibitors, such as anti-IgE antibodies, such as omalizumab) or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinib, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738); and / or (4) treatment with one or more BTK inhibitors (e.g., ribrutinib and / or rizabrutinib). In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to the following treatments: (1) antihistamine treatment (e.g., H1- and / or H2-antihistamine treatment); and (2) treatment with one or more immunomodulatory agents or anti-inflammatory agents (e.g., IgE inhibitors, e.g., anti-IgE antibodies, e.g., omalizumab). or ligralizumab; IL-4R inhibitors, such as anti-IL-4R antibodies, such as dupilumab IL-5R inhibitors, such as anti-IL-5R antibodies, such as brexlizumab IL-5 inhibitors, such as anti-IL-5 antibodies, such as mepolizumab; Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, such as lerilizumab; TSLP or TSLPR inhibitors, such as anti-TSLP antibodies or anti-TSLPR antibodies, such as tezemab; TM ); C5aR inhibitors, e.g., anti-C5aR antibodies, e.g., idolinimab, and / or CD200R inhibitors, e.g., anti-CD200R antibodies, e.g., LY3454738). In certain embodiments, the mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to the following treatments: (1) antihistamine treatment (e.g., H1- and / or H2-antihistamine treatment); (2) treatment with an IgE inhibitor, e.g., an anti-IgE antibody, e.g., omalizumab or ligralizumab; and (3) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to: (1) antihistamine therapy (e.g., H1- and / or H2-antihistamine therapy); and (2) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is refractory to and resistant to: (1) treatment with an IgE inhibitor, such as an anti-IgE antibody, such as omalizumab or ligralizumab; and (2) treatment with an IL-4R inhibitor, such as an anti-IL-4R antibody, such as dupilumab In certain embodiments, the mast cell-associated disorder is chronic prurigo and is treated with an IL-4R inhibitor (e.g., an anti-IL-4R antibody, e.g., dupilumab). ) treatment is refractory and resistant to said treatment. In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory and resistant to treatment with an IL-31 receptor alpha inhibitor (e.g., an anti-IL-31 receptor alpha antibody, such as nemolizumab). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory and resistant to treatment with a Janus kinase 1 inhibitor (e.g., INCB054707 or abrocitinib). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory and resistant to treatment with a neurokinin-1 (NK1) receptor antagonist (e.g., serlopitant). In certain embodiments, the mast cell-related disorder is chronic prurigo and is refractory and resistant to treatment with a PDE4 and / or TNF-alpha inhibitor (e.g., apremilast). In certain embodiments, the mast cell-associated disorder is chronic prurigo and is refractory and resistant to treatment with an OSMRβ inhibitor (e.g., an anti-OSMRβ antibody, such as vesalizumab). In certain embodiments, the mast cell-associated disorder is chronic prurigo and is refractory and resistant to treatment with one, two, three or more of the above-mentioned treatments for chronic prurigo.

[0461] In various embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are recurrent conditions that recur after one or more previous treatments for this disease.In certain embodiments, one or more previous treatments include at least one standard care therapy for this disease.In certain embodiments, one or more previous treatments are all standard care therapy for this disease.In certain embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are recurrent conditions that recur after antihistamine treatment.In certain embodiments, mast cell related conditions (such as urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or eosinophil related conditions (such as eosinophilic esophagitis) are recurrent conditions that recur after H1-antihistamine treatment. In certain embodiments, a mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) is a recurrent disorder that recurs after treatment with an H2-antihistamine. In certain embodiments, a mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) is a recurrent disorder that recurs after treatment with an H1- and H2-antihistamine. In certain embodiments, a mast cell-associated disorder (e.g., urticaria, e.g., chronic induced urticaria or chronic spontaneous urticaria) or an eosinophil-associated disorder (e.g., eosinophilic esophagitis) is a recurrent disorder that recurs after treatment with one or more leukotriene receptor antagonists. In certain embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is a recurrent disorder that relapses after treatment with one or more immunomodulatory agents or anti-inflammatory agents. In specific embodiments, the mast cell-associated disorder (e.g., urticaria, such as chronic induced urticaria or chronic spontaneous urticaria) or the eosinophil-associated disorder (e.g., eosinophilic esophagitis) is a recur...

Claims

1. A pharmaceutical composition comprising: (i) an antibody or an antigen-binding fragment thereof that immunospecifically binds to human KIT; (ii) a buffer; (iii) a salt; and (iv) an excipient.

2. The pharmaceutical composition of claim 1, having a pH of about 4 to about 7.

3. The pharmaceutical composition of claim 2, having a pH of about 5 to about 6.

4. The pharmaceutical composition of claim 3, having a pH of about 5.

5.

5. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the salt is an alkali metal salt.

6. The pharmaceutical composition of claim 5, wherein the alkali metal salt is sodium chloride.

7. The pharmaceutical composition of claim 6, wherein the concentration of sodium chloride is about 25 mM to about 100 mM.

8. The pharmaceutical composition of claim 7, wherein the concentration of sodium chloride is about 50 mM.

9. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the buffer is an alkali metal acetate.

10. The pharmaceutical composition of claim 9, wherein the alkali metal acetate is sodium acetate.

11. The pharmaceutical composition of claim 10, wherein the concentration of sodium acetate is about 1 mM to about 50 mM.

12. The pharmaceutical composition of claim 11, wherein the concentration of sodium acetate is about 25 mM.

13. The pharmaceutical composition of any one of the preceding claims, wherein the excipient is a sugar, a sugar alcohol, an amino acid, or any combination thereof.

14. The pharmaceutical composition of claim 13, wherein the excipient is mannitol, sorbitol, sucrose, trehalose, glycine, arginine, alanine, histidine, or any combination thereof.

15. The pharmaceutical composition of claim 13, wherein the excipient is mannitol, sorbitol, sucrose, trehalose, glycine, arginine, histidine, or any combination thereof.

16. The pharmaceutical composition of claim 14 or 15, wherein the excipient is mannitol, sucrose, arginine, histidine, or any combination thereof.

17. The pharmaceutical composition of any one of claims 13-16, wherein the excipient is mannitol.

18. The pharmaceutical composition of claim 17, wherein the concentration of mannitol is about 1% to about 10%.

19. The pharmaceutical composition of claim 18, wherein the concentration of mannitol is about 3%.

20. The pharmaceutical composition of any one of the preceding claims, wherein the concentration of the antibody or antigen-binding fragment thereof is about 50 mg / ml to about 500 mg / ml.

21. The pharmaceutical composition of claim 20, wherein the concentration of the antibody or antigen-binding fragment thereof is about 100 mg / ml to about 400 mg / ml.

22. The pharmaceutical composition of claim 21, wherein the concentration of the antibody or antigen-binding fragment thereof is about 150 mg / ml.

23. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 50 mg / ml to about 500 mg / ml; (ii) an alkali metal acetate at a concentration of about 1 mM to about 50 mM; (iii) an alkali metal chloride at a concentration of about 25 mM to about 100 mM; and (iv) mannitol at a concentration of about 1% to about 10%; wherein the pH of the pharmaceutical composition is about 5 to about 6.

24. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 50 mg / ml to about 500 mg / ml; (ii) sodium acetate at a concentration of about 1 mM to about 50 mM; (iii) sodium chloride at a concentration of about 25 mM to about 100 mM; and (iv) mannitol at a concentration of about 1% to about 10%; wherein the pH of the pharmaceutical composition is about 5 to about 6.

25. A pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT at a concentration of about 150 mg / ml; (ii) sodium acetate at a concentration of about 25 mM; (iii) sodium chloride at a concentration of about 50 mM; and (iv) mannitol at a concentration of about 3%; wherein the pH of the pharmaceutical composition is about 5.

5.

26. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises: (A)(i) a light chain variable region ("VL") comprising VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; and (ii) a heavy chain variable region ("VH"), said VH comprising VH CDR1, VH CDR2 and VH CDR3, said VH CDR1, VH CDR2 and VH CDR3 comprising 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, wherein the VL CDR1, VL CDR2, and VL CDR3 comprise 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, wherein the VH CDR1, the VH CDR2 and the VH CDR3 comprise 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, wherein the VL CDR1, VL CDR2, and VL CDR3 comprise 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, wherein the VH CDR1, the VH CDR2 and the VH CDR3 comprise 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, wherein the VL CDR1, VL CDR2, and VL CDR3 comprise 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, wherein the VH CDR1, VH CDR2 and VH CDR3 comprise 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, wherein the VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively; and (ii) VH, comprising VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

27. A pharmaceutical composition as described in any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises: a VL, wherein the VL comprises VL CDRs 1-3, wherein the VL CDRs 1-3 comprise the amino acid sequences of SEQ ID NOs: 2-4, respectively; and a VH, wherein the VH comprises VH CDRs 1-3, wherein the VH CDRs 1-3 comprise the amino acid sequences of SEQ ID NOs: 5-7, respectively.

28. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises (i) a VL comprising the following amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X K 1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aliphatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 is an amino acid with an aliphatic hydroxyl side chain or P, X K5 is an amino acid with a charged or acidic side chain, and X K6 is an amino acid with an aromatic side chain; and (ii) a VH comprising the following amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAPGKGLEWIARIYPGSGNTYYNEKFKGRX H 4TX H5 TAX H6 KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X H 1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid having an acidic or amide derivative side chain, and X H8 It is an amino acid with an aliphatic hydroxyl side chain.

29. The pharmaceutical composition of claim 28, wherein X K1 is amino acid F or S, X K2 is amino acid A or S, X K3 is amino acid T or S, X K4 is amino acid S or P, X K5 is amino acid D or T, X K6 is amino acid F or Y, X H1 is amino acid L or V, X H2 is amino acid L or V, X H3 is amino acid K or R, X H4 is amino acid V or A, X H5 is amino acid L or I, X H6 is amino acid E or D, X H7 is amino acid Q or E, and X H8 It is the amino acid S or T.

30. A pharmaceutical composition as described in any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15 and 16; and a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11 and 12.

31. The pharmaceutical composition of any of the preceding claims, wherein the antibody comprises a human heavy chain constant region, and wherein the human heavy chain constant region is a human IgG1 constant region.

32. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the antibody comprises a modified human Fc region or domain.

33. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the antibody comprises a modified human IgG1 Fc region or domain.

34. The pharmaceutical composition of claim 33, wherein the modified human IgGl Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q and 322Q numbered according to the EU index as set forth in Kabat.

35. The pharmaceutical composition of claim 34, wherein the modified human IgGl Fc region or domain further comprises the non-naturally occurring amino acids 252Y, 254T and 256E numbered according to the EU index as set forth in Kabat.

36. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the antibody comprises: (i) VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q and 322Q, numbered according to the EU index as set forth in Kabat.

37. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the antibody comprises: (i) VL comprising the amino acid sequence of SEQ ID NO: 14; (ii) VH comprising the amino acid sequence of SEQ ID NO: 10; and (iii) a modified human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E, numbered according to the EU index as set forth in Kabat.

38. A pharmaceutical composition as described in any of the preceding claims, wherein the antibody comprises a heavy chain comprising the following amino acid sequence:

39. A pharmaceutical composition as described in any of the preceding claims, wherein the antibody comprises a light chain comprising the following amino acid sequence:

40. A pharmaceutical kit comprising the pharmaceutical composition of any preceding claim.

41. A method for preventing, treating or managing a KIT-related disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 1-39.

42. The method of claim 41, wherein the pharmaceutical composition is administered subcutaneously to the subject.

43. The method of claim 41 or 42, wherein the KIT-associated disorder is a mast cell-associated disorder, an eosinophil-associated disorder, cancer, asthma, an inflammatory disorder, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.

44. The method of claim 43, wherein the KIT-associated disorder is a mast cell-associated disorder.

45. The method of claim 43, wherein the KIT-associated disorder is an eosinophil-associated disorder.

46. ​​The method of any one of claims 41-45, further comprising administering to the subject a second therapeutic agent.

47. The method of claim 46, 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.

48. The method of any one of claims 41-47, wherein the subject is a human.

49. The method of any one of claims 41-48, wherein the subject is administered ≥ 1.5 mg / kg of the antibody or antigen-binding fragment thereof per dose.

50. The method of any one of claims 41-48, wherein about 1.5 mg / kg to about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

51. The method of any one of claims 41-48, wherein about 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

52. The method of any one of claims 41-48, wherein about 3.0 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

53. The method of any one of claims 41-48, wherein about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject per dose.

54. The method of any one of claims 41-53, wherein two doses of the antibody or antigen-binding fragment thereof are administered to the subject.

55. The method of any one of claims 41-53, wherein three doses of the antibody or antigen-binding fragment thereof are administered to the subject.

56. The method of any one of claims 41-55, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 4 weeks.

57. The method of any one of claims 41-55, wherein the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks.

58. The method of any one of claims 41-48, wherein about 1.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every four weeks for three doses.

59. The method of any one of claims 41-48, wherein about 3.0 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks for two doses.

60. The method of any one of claims 41-48, wherein about 4.5 mg / kg of the antibody or antigen-binding fragment thereof is administered to the subject once every 8 weeks for two doses.

61. A method for preparing the pharmaceutical composition of any one of claims 1-39, comprising combining the antibody or antigen-binding fragment thereof with the buffer, the salt, and the excipient.

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