CN117136070A

CN117136070A - anti-KIT antibodies and uses thereof

Info

Publication number: CN117136070A
Application number: CN202280023490.XA
Authority: CN
Inventors: J·戈尔茨坦
Original assignee: Celldex Therapeutics Inc
Current assignee: Celldex Therapeutics Inc
Priority date: 2021-01-22
Filing date: 2022-01-21
Publication date: 2023-11-28

Abstract

Provided herein are antibodies that immunospecifically bind to KIT (receptor tyrosine kinase) and uses thereof. Polynucleotides and vectors encoding such antibodies, cells comprising such polynucleotides or vectors, and methods of making such antibodies are also provided.

Description

anti-KIT antibodies and uses thereof

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional patent application Ser. No.63/140,642, filed on 22. 1/2021, and Ser. No. 63/238,649, filed on 30. 8/2021, which are incorporated herein by reference in their entirety.

Reference to an electronically submitted sequence Listing

The present application incorporates by reference the sequence listing filed with the present application as a text file entitled "seqlipsting_12638-170-228. Txt" created at month 1, 10 of 2022 and having a size of 44,171 bytes.

1. Technical field

2. Background art

KIT (or c-KIT) is a type III receptor tyrosine kinase encoded by the c-KIT gene. KIT comprises 5 extracellular immunoglobulin (Ig) -like domains separated by a kinase insert, a single transmembrane region, an inhibitory cytoplasmic membrane-proximal domain, and a cytokinin kinase domain (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 has been cloned as described in Yarden et al, EMBO J.,1987, 6:3341-3351. KIT is also known as CD117 or stem cell factor receptor ("SCFR") because it is a receptor for stem cell factor ("SCF") ligands (also known as Steel factor or KIT ligand). SCF ligands that bind to the first three extracellular Ig-like domains of KIT cause receptor dimerization and thereby activate endogenous 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). Stat, src, ERK and AKT signaling pathway members have been shown to be downstream signaling proteins of KIT signaling.

The fourth (D4) and fifth (D5) extracellular Ig-like domains of KIT are believed to mediate receptor dimerization (see, e.g., international patent application publication No. WO 2008/153926; yuzawa et al, cell,2007, 130:323-334).

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., prism, 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).

Antibodies that immunospecifically bind to human KIT are known, for example, from international patent publication No WO2014018625A1, the disclosure of which is incorporated herein by reference in its entirety.

There is a need to provide improved antibodies against human KIT.

3. Brief description of the invention

In one aspect, provided herein is an antibody that immunospecifically binds to human KIT comprising: (i) A light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; (ii) A heavy chain variable region ("VH") comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by EU numbering as set forth in Kabat. In particular embodiments, the modified (e.g., mutated) human IgG1 Fc region or domain further comprises non-naturally occurring amino acids 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat.

In specific embodiments, (i) the VL of the antibody comprises the amino acid sequence: DIVMTQSPSX _K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX _K2 LIYSASYR YSGVPDRFX _K3 GSGSGTDFTLTISSLQX _K4 EDFAX _K5 YX _K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X _K1 Is an amino acid having an aromatic or aliphatic hydroxyl side chain, X _K2 Is an amino acid having an aliphatic or aliphatic hydroxyl side chain, X _K3 Is a toolAmino acids having aliphatic hydroxy side chains, X _K4 Is an amino acid with an aliphatic hydroxyl side chain or P, X _K5 Is an amino acid having a charged or acidic side chain and X _K6 Is an amino acid having an aromatic side chain; and (ii) the VH of the antibody comprises the amino acid sequence: QVQLVQSGAEX _H1 KKPGASVKX _H2 SCKASGYTFTDYYINWVX _H3 QAPGKGLEWIARIY PGSGNTYYNEKFKGRX _H4 TX _H5 TAX _H6 KSTSTAYMX _H7 LSSLRSEDX _H ₈ AVYFCARGVYY FDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X _H1 Is an amino acid having an aliphatic side chain, X _H2 Is an amino acid having an aliphatic side chain, X _H3 Is an amino acid having a polar or basic side chain, X _H4 Is an amino acid having an aliphatic side chain, X _H5 Is an amino acid having an aliphatic side chain, X _H6 Is an amino acid with an acidic side chain, X _H7 Is an amino acid having acidic and amide derivative side chains, and X _H8 Is an amino acid having 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 the amino acid T or S, X _K4 Is the 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 the 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 Is amino acid S or T.

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

In particular embodiments, the antibody comprises: (i) a polypeptide comprising SEQ ID NO:14, VL of the amino acid sequence shown in seq id no; (ii) a polypeptide comprising SEQ ID NO:10, VH of the amino acid sequence shown in fig; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by EU numbering as set forth in Kabat.

In particular embodiments, the antibody comprises: (i) a polypeptide comprising SEQ ID NO:14, VL of the amino acid sequence shown in seq id no; (ii) a polypeptide comprising SEQ ID NO:10, VH of the amino acid sequence shown in fig; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat.

In particular embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYINWVRQAPGKGLEWIARIYPGSGNTYYNEKFKGRATLTADKSTSTAYMQLSSLRSEDTAVYFCARGVYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21).

In particular embodiments, the antibody comprises: a light chain comprising the amino acid sequence: DIVMTQSPSSLSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

In specific embodiments, the antibody comprises (i) a heavy chain comprising the amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYINWVRQAPGKGLEWIARIYPGSGNTYYNEKFKGRATLTADKSTSTAYMQLSSLRSEDTAVYFCARGVYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21); and (ii) a light chain comprising the amino acid sequence: DIVMTQSPSSLSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

In another aspect, provided herein are conjugates comprising an antibody provided herein linked to an agent.

In another aspect, provided herein are pharmaceutical compositions comprising an antibody or conjugate provided herein and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a polynucleotide or combination of polynucleotides comprising nucleotide sequences encoding an antibody provided herein or VH and VL of the antibody.

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

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

In another aspect, provided herein are kits comprising an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein.

In another aspect, provided herein is a method for protecting, treating, or controlling a KIT-related disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein. In particular embodiments, the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In particular embodiments, the KIT-related disorder is a mast cell-related disorder. In a specific embodiment, the mast cell related disorder is chronic urticaria. In one embodiment, the chronic urticaria is chronic induced urticaria. In one embodiment, the chronic inductive urticaria is cold urticaria. In one embodiment, the chronic induced urticaria is a symptomatic skin scarification. In one embodiment, the chronic inductive urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic idiopathic urticaria. In particular embodiments, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

In certain embodiments, the methods provided herein further comprise administering a second therapeutic agent to the subject. In particular 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.

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

In another aspect, provided herein is an in vitro method for diagnosing a subject as having a KIT-related disorder, wherein the method comprises contacting a cell or sample obtained from the subject with an antibody provided herein and detecting the expression level of KIT in the cell or sample.

In another aspect, provided herein are methods of producing an antibody, wherein the methods comprise culturing a host cell provided herein and/or expressing the antibody using a host cell provided herein. In particular embodiments, the method further comprises purifying antibodies obtained from the host cell.

In another aspect, provided herein is the use of an antibody provided herein, a conjugate provided herein, or a pharmaceutical composition provided herein for the manufacture of a medicament for protecting, treating, or controlling a KIT-related disorder. In particular embodiments, the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In particular embodiments, the KIT-related disorder is a mast cell-related disorder. In a specific embodiment, the mast cell related disorder is chronic urticaria. In one embodiment, the chronic urticaria is chronic induced urticaria. In one embodiment, the chronic inductive urticaria is cold urticaria. In one embodiment, the chronic induced urticaria is a symptomatic skin scarification. In one embodiment, the chronic inductive urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic idiopathic urticaria. In particular embodiments, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

In certain embodiments, the agents described herein are produced for administration in combination with a second therapeutic agent. In particular 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.

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

In another aspect, provided herein are antibodies described herein, conjugates described herein, or pharmaceutical compositions described herein for use in a method of protecting, treating, or controlling a KIT-related disorder. In particular embodiments, the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In particular embodiments, the KIT-related disorder is a mast cell-related disorder. In a specific embodiment, the mast cell related disorder is chronic urticaria. In one embodiment, the chronic urticaria is chronic induced urticaria. In one embodiment, the chronic inductive urticaria is cold urticaria. In one embodiment, the chronic induced urticaria is a symptomatic skin scarification. In one embodiment, the chronic inductive urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic idiopathic urticaria. In particular embodiments, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

In certain embodiments, the method further comprises administering a second therapeutic agent to the subject. In particular 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.

In another aspect, provided herein are antibodies described herein, conjugates described herein, or pharmaceutical compositions described herein for use in a method of inhibiting KIT activity in a cell expressing KIT. In particular embodiments, the method inhibits KIT activity in a cell expressing KIT by at least about 10%.

In another aspect, provided herein is a method of protecting, treating, or managing chronic urticaria in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (1) an antibody that immunospecifically binds to human KIT or an antigen-binding fragment thereof, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the conjugate and a pharmaceutically acceptable carrier. In certain embodiments, the human KIT comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. In certain embodiments, the antibody specifically binds to the D4 or D5 region of human KIT. In certain embodiments, the antibodies comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human Fc region or domain). In certain embodiments, the antibody has reduced Fc receptor binding activity (specifically reduced FcrR binding activity). In certain embodiments, the antibody does not cause significant degranulation of FcgRI-expressing human mast cells. In certain embodiments, the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity. In certain embodiments, the antibody comprises: (A) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; (B) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:26 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; (C) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 28. SEQ ID NO:29 and SEQ ID NO:30, an amino acid sequence shown in seq id no; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:31 and SEQ ID NO:32, an amino acid sequence shown in seq id no; (D) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 33. SEQ ID NO:34 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; or (E) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 35. SEQ ID NO:36 and SEQ ID NO:37, and a nucleotide sequence shown in seq id no; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 38. SEQ ID NO:39 and SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no. In one embodiment, the chronic urticaria is chronic induced urticaria. In one embodiment, the chronic inductive urticaria is cold urticaria. In one embodiment, the chronic induced urticaria is a symptomatic skin scarification. In one embodiment, the chronic inductive urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic idiopathic urticaria.

In another aspect, provided herein is the use of (1) an antibody that immunospecifically binds to human KIT or an antigen-binding fragment thereof, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the conjugate and a pharmaceutically acceptable carrier, for the manufacture of a medicament for the protection, treatment, or management of chronic urticaria in a subject. In certain embodiments, the human KIT comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. In certain embodiments, the antibody specifically binds to the D4 or D5 region of human KIT. In certain embodiments, the antibodies comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human Fc region or domain). In certain embodiments, the antibody has reduced Fc receptor binding activity (specifically reduced FcrR binding activity). In certain embodiments, the antibody does not cause significant degranulation of FcgRI-expressing human mast cells. In certain embodiments, the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity. In certain embodiments, the antibody comprises: (A) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; (B) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:26 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; (C) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 28. SEQ ID NO:29 and SEQ ID NO:30, an amino acid sequence shown in seq id no; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:31 and SEQ ID NO:32, an amino acid sequence shown in seq id no; (D) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 33. SEQ ID NO:34 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; or (E) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 35. SEQ ID NO:36 and SEQ ID NO:37, and a nucleotide sequence shown in seq id no; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 38. SEQ ID NO:39 and SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no. In a specific embodiment, the chronic urticaria is chronic induced urticaria. In one embodiment, the chronic inductive urticaria is cold urticaria. In one embodiment, the chronic induced urticaria is a symptomatic skin scarification. In one embodiment, the chronic inductive urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic idiopathic urticaria.

In another aspect, provided herein are (1) antibodies that immunospecifically bind to human KIT or an antigen-binding fragment thereof, (2) conjugates comprising the antibodies or antigen-binding fragments thereof linked to an agent, or (3) pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, or the conjugates and a pharmaceutically acceptable carrier, for use in a method of protecting, treating, or managing chronic urticaria. In certain embodiments, the human KIT comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. In certain embodiments, the antibody specifically binds to the D4 or D5 region of human KIT. In certain embodiments, the antibodies comprise a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human Fc region or domain). In certain embodiments, the antibody has reduced Fc receptor binding activity (specifically reduced FcrR binding activity). In certain embodiments, the antibody does not cause significant degranulation of FcgRI-expressing human mast cells. In certain embodiments, the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity. In certain embodiments, the antibody comprises: (A) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; (B) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:26 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; (C) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 28. SEQ ID NO:29 and SEQ ID NO:30, an amino acid sequence shown in seq id no; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:31 and SEQ ID NO:32, an amino acid sequence shown in seq id no; (D) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 33. SEQ ID NO:34 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; or (E) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 35. SEQ ID NO:36 and SEQ ID NO:37, and a nucleotide sequence shown in seq id no; and (ii) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 38. SEQ ID NO:39 and SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no. In a specific embodiment, the chronic urticaria is chronic induced urticaria. In one embodiment, the chronic inductive urticaria is cold urticaria. In one embodiment, the chronic induced urticaria is a symptomatic skin scarification. In one embodiment, the chronic inductive urticaria is cholinergic urticaria. In another embodiment, the chronic urticaria is chronic idiopathic urticaria.

3.1 illustrative embodiments

1. An antibody that immunospecifically binds to human KIT comprising:

(i) A light chain variable region ("VL") comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure;

(ii) A heavy chain variable region ("VH") comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; and

(iii) A modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by EU numbering as set forth in Kabat.

2. The antibody of embodiment 1, wherein the modified (e.g., mutated) human IgG1 Fc region or domain further comprises non-naturally occurring amino acids 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat.

3. The antibody according to embodiment 1 or 2, wherein:

(i) The VL comprises the amino acid sequence:

DIVMTQSPSX _K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX _K2 LIYSAS YRYSGVPDRFX _K3 GSGSGTDFTLTISSLQX _K4 EDFAX _K5 YX _K6 CQQYNSYPRTFGGGTKVE IK (SEQ ID NO: 17), wherein X _K1 Is an amino acid having an aromatic or aliphatic hydroxyl side chain, X _K2 Is an amino acid having an aliphatic or aliphatic hydroxyl side chain, X _K3 Is an amino acid having 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 having a charged or acidic side chain and X _K6 Is an amino acid having an aromatic side chain; and

(ii) The VH comprises the amino acid sequence:

QVQLVQSGAEX _H1 KKPGASVKX _H2 SCKASGYTFTDYYINWVX _H3 QAPGKGLEWIA RIYPGSGNTYYNEKFKGRX _H4 TX _H5 TAX _H6 KSTSTAYMX _H7 LSSLRSEDX _H8 AVYFCARGV YYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X _H1 Is an amino acid having an aliphatic side chain, X _H2 Is an amino acid having an aliphatic side chain, X _H3 Is an amino acid having a polar or basic side chain, X _H4 Is an amino acid having an aliphatic side chain, X _H5 Is an amino acid having an aliphatic side chain, X _H6 Is an amino acid with an acidic side chain, X _H7 Is an amino acid having acidic and amide derivative side chains, and X _H8 Is an amino acid having an aliphatic hydroxyl side chain.

4. The antibody of embodiment 3, wherein X _K1 Is amino acid F or S, X _K2 Is amino acid A or S, X _K3 Is the amino acid T or S, X _K4 Is the 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 the 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 Is amino acid S or T.

5. The antibody of any one of embodiments 1 to 4, wherein:

i) Comprising SEQ ID NO: 13. 14, 15 or 16, and

ii) a polypeptide comprising SEQ ID NO: 8. 9, 10, 11 or 12.

6. The antibody of any one of embodiments 1 to 5, comprising:

(i) Comprising SEQ ID NO:14, VL of the amino acid sequence shown in seq id no;

(ii) Comprising SEQ ID NO:10, VH of the amino acid sequence shown in fig; and

7. The antibody of any one of embodiments 1 to 5, comprising:

(i) Comprising SEQ ID NO:14, VL of the amino acid sequence shown in seq id no;

(ii) Comprising SEQ ID NO:10, VH of the amino acid sequence shown in fig; 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 as numbered by EU numbering as set forth in Kabat.

8. The antibody of any one of embodiments 1 to 5, comprising: a heavy chain comprising the amino acid sequence:

QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYINWVRQAPGKGLEWIARIYPGSGNTYYNEKFKGRATLTADKSTSTAYMQLSSLRSEDTAVYFCARGVYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO：21)。

9. the antibody of any one of embodiments 1 to 5, comprising: a light chain comprising the amino acid sequence:

DIVMTQSPSSLSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：22)。

10. the antibody of any one of embodiments 1 to 5, comprising: a heavy chain comprising the amino acid sequence:

QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYINWVRQAPGKGLEWIARIYPGSGNTYYNEKFKGRATLTADKSTSTAYMQLSSLRSEDTAVYFCARGVYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21); and a light chain comprising the amino acid sequence:

11. a conjugate comprising the antibody of any one of embodiments 1 to 10 linked to a reagent.

12. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 10 or the conjugate of embodiment 11, and a pharmaceutically acceptable carrier.

13. A polynucleotide or combination of polynucleotides comprising a nucleotide sequence encoding an antibody according to any one of embodiments 1 to 10 or a VH and VL of said antibody.

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

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

16. A kit comprising an antibody according to any one of embodiments 1 to 10, a conjugate according to embodiment 11 or a pharmaceutical composition according to embodiment 12.

17. A method of protecting, treating or controlling a KIT-associated disorder comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to any one of embodiments 1-10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12.

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

19. The method of embodiment 18, wherein the KIT-related disorder is a mast cell-related disorder.

20. The method of embodiment 19, wherein the mast cell-related disorder is chronic urticaria.

21. The method of embodiment 20, wherein the chronic urticaria is chronic-induced urticaria.

22. The method of embodiment 21, wherein the chronic-induced urticaria is cold urticaria.

23. The method of embodiment 21, wherein the chronic-induced urticaria is symptomatic skin scarification.

24. The method of embodiment 21, wherein the chronic-induced urticaria is cholinergic urticaria.

25. The method of embodiment 20, wherein the chronic urticaria is chronic idiopathic urticaria.

26. The method of embodiment 18, wherein the KIT-related disorder is an eosinophil-related disorder.

27. The method of any one of embodiments 17-26, further comprising administering a second therapeutic agent to the subject.

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

29. A method for inhibiting KIT activity in a cell expressing KIT, the method comprising contacting the cell with an effective amount of an antibody according to any one of embodiments 1-10, a conjugate according to embodiment 11, or a pharmaceutical composition according to embodiment 12.

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

31. An in vitro method for diagnosing a subject as suffering from a KIT-related disorder, wherein the method comprises contacting a cell or sample obtained from the subject with an antibody according to any one of embodiments 1 to 10 and detecting the expression level of KIT in the cell or sample.

32. A method of producing an antibody, wherein the method comprises culturing the host cell of embodiment 15 and/or expressing the antibody using the host cell of embodiment 15.

33. The method of embodiment 32, further comprising purifying antibodies obtained from the host cell.

34. The use of the antibody of any one of embodiments 1-10, for the manufacture of a medicament for protecting, treating or controlling a KIT-related disorder, the conjugate of embodiment 11 or the pharmaceutical composition of embodiment 12.

35. The use of embodiment 34, wherein the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis.

36. The use of embodiment 35, wherein the KIT-related disorder is a mast cell-related disorder.

37. The use of embodiment 36, wherein the mast cell-related disorder is chronic urticaria.

38. The use of embodiment 37, wherein the chronic urticaria is chronic inducible urticaria.

39. The use of embodiment 38, wherein the chronic inducible urticaria is cold urticaria.

40. The use of embodiment 38, wherein the chronic-induced urticaria is a symptomatic skin scarification.

41. The use according to embodiment 38, wherein the chronic inductive urticaria is cholinergic urticaria.

42. The use of embodiment 37, wherein the chronic urticaria is chronic idiopathic urticaria.

43. The use of embodiment 35, wherein the KIT-related disorder is an eosinophil-related disorder.

44. The use of any one of embodiments 34 to 43, wherein the medicament is produced for administration in combination with a second therapeutic agent.

45. The use of embodiment 44, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulator, or an anti-inflammatory agent.

46. The use of the antibody of any one of embodiments 1-10, for the manufacture of a medicament for inhibiting the activity of KIT in a KIT expressing cell, the conjugate of embodiment 11 or the pharmaceutical composition of embodiment 12.

47. The use of embodiment 46, wherein the agent inhibits KIT activity in a cell expressing KIT by at least about 10%.

48. The antibody of any one of embodiments 1 to 10, the conjugate according to embodiment 11 or the pharmaceutical composition according to embodiment 12 for use in a method of protecting, treating or controlling a KIT-related disorder.

49. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 48, wherein the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal function disorder, or fibrosis.

50. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 49, wherein the KIT-associated disorder is a mast cell-associated disorder.

51. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 50, wherein the mast cell-related disorder is chronic urticaria.

52. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 51, wherein the chronic urticaria is chronic inducible urticaria.

53. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 52, wherein the chronically induced urticaria is cold urticaria.

54. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 52, wherein the chronic induced urticaria is symptomatic skin scarification.

55. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 52, wherein the chronically induced urticaria is cholinergic urticaria.

56. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 51, wherein the chronic urticaria is chronic idiopathic urticaria.

57. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 49, wherein the KIT-associated disorder is an eosinophil-associated disorder.

58. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 48-57, wherein the method further comprises administering a second therapeutic agent to the subject.

59. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 58, wherein the second therapeutic agent is a chemotherapeutic agent, histone deacetylase inhibitor, antibody, cytokine, tyrosine kinase inhibitor, antihistamine, leukotriene receptor antagonist, immunomodulator, or anti-inflammatory agent.

60. The antibody of any one of embodiments 1 to 10, the conjugate according to embodiment 11 or the pharmaceutical composition according to embodiment 12 for use in a method for inhibiting the activity of KIT in a cell expressing KIT.

61. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 60, wherein the method inhibits KIT activity in a KIT-expressing cell by at least about 10%.

62. A method for protecting, treating, or controlling chronic urticaria in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharmaceutically acceptable carrier.

63. The method of embodiment 62, wherein the human KIT comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

64. The method of embodiment 62 or 63, wherein the antibody specifically binds to the D4 or D5 region of human KIT.

65. The method of any one of embodiments 62 to 64, wherein the antibody comprises a modified (e.g., mutated) Fc region or domain.

66. The method of any one of embodiments 62-65, wherein the antibody comprises a modified (e.g., mutated) human Fc region or domain.

67. The method of any one of embodiments 62 to 66, wherein the antibody has reduced Fc receptor binding activity (specifically reduced FcrR binding activity).

68. The method of any one of embodiments 62 to 67, wherein the antibody does not cause significant degranulation of FcgRI-expressing human mast cells.

69. The method of any one of embodiments 62-68, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.

70. The method of any one of embodiments 62 to 69, wherein the antibody comprises:

(A) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and

(ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7;

(B) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and

(ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:26 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no;

(C) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 28. SEQ ID NO:29 and SEQ ID NO:30, an amino acid sequence shown in seq id no; and

(ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 25. SEQ ID NO:31 and SEQ ID NO:32, an amino acid sequence shown in seq id no;

(D) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide sequence shown in the figure; and

(ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 33. SEQ ID NO:34 and SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no; or (b)

(E) (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 35. SEQ ID NO:36 and SEQ ID NO:37, and a nucleotide sequence shown in seq id no; and

(ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 38. SEQ ID NO:39 and SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no.

71. The method of embodiment 70, wherein the chronic urticaria is chronic-induced urticaria.

72. The method of embodiment 71, wherein the chronic-induced urticaria is cold urticaria.

73. The method of embodiment 71, wherein the chronic-induced urticaria is symptomatic skin scarification.

74. The method of embodiment 71, wherein the chronic-induced urticaria is cholinergic urticaria.

75. The method of embodiment 70, wherein the chronic urticaria is chronic idiopathic urticaria.

76. The method of any one of embodiments 70-75, further comprising administering to the subject a second therapeutic agent.

77. The method of embodiment 76, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulator, or an anti-inflammatory agent.

78. Use of (1) an antibody or antigen-binding fragment thereof that immunospecifically binds to human KIT, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharmaceutically acceptable carrier, for the manufacture of a medicament for protecting, treating or controlling chronic urticaria in a subject.

79. The use of embodiment 78, wherein the human KIT comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

80. The use of embodiments 78 or 79, wherein the antibody specifically binds to the D4 or D5 region of human KIT.

81. The use of any one of embodiments 78 to 80, wherein the antibody comprises a modified (e.g., mutated) Fc region or domain.

82. The use of any one of embodiments 78 to 81, wherein the antibody comprises a modified (e.g., mutated) human Fc region or domain.

83. The use of any one of embodiments 78 to 82, wherein the antibody has reduced Fc receptor binding activity (specifically reduced FcrR binding activity).

84. The use according to any one of embodiments 78 to 83, wherein the antibody does not cause significant degranulation of FcgRI-expressing human mast cells.

85. The use of any one of embodiments 78 to 84, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.

86. The use of any one of embodiments 78 to 85, wherein the antibody comprises:

87. The use of embodiment 86, wherein the chronic urticaria is chronic inducible urticaria.

88. The use of embodiment 87, wherein the chronic-induction urticaria is cold urticaria.

89. The use of embodiment 87, wherein the chronic-induced urticaria is a symptomatic skin scarification.

90. The use of embodiment 87, wherein the chronic-induced urticaria is cholinergic urticaria.

91. The use of embodiment 86, wherein the chronic urticaria is chronic idiopathic urticaria.

92. The use of any one of embodiments 86-91, wherein the agent is produced for administration in combination with a second therapeutic agent.

93. The use of embodiment 92, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulator, or an anti-inflammatory agent.

94. An antibody that immunospecifically binds to human KIT or an antigen-binding fragment thereof, a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or the conjugate and a pharmaceutically acceptable carrier for use in a method of protecting, treating or controlling chronic urticaria in a subject.

95. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 94, wherein the human KIT comprises the amino acid sequence of SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

96. An antibody, conjugate, or pharmaceutical composition for use according to embodiments 94 or 95, wherein the antibody specifically binds to the D4 or D5 region of human KIT.

97. An antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94-96, wherein the antibody comprises a modified (e.g., mutated) Fc region or domain.

98. An antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94-97, wherein the antibody comprises a modified (e.g., mutated) human Fc region or domain.

99. An antibody, conjugate or pharmaceutical composition for use according to any one of embodiments 94 to 98, wherein the antibody has reduced Fc receptor binding activity (in particular reduced FcrR binding activity).

100. An antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94 to 99, wherein the antibody does not cause significant degranulation of FcgRI-expressing human mast cells.

101. An antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 94-100, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.

102. An antibody, conjugate or pharmaceutical composition for use according to any one of embodiments 94 to 101, wherein the antibody comprises:

103. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 102, wherein the chronic urticaria is chronic inducible urticaria.

104. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 103, wherein the chronic induced urticaria is cold urticaria.

105. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 103, wherein the chronic induced urticaria is symptomatic skin scarification.

106. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 103, wherein the chronic inductive urticaria is cholinergic urticaria.

107. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 102, wherein the chronic urticaria is chronic idiopathic urticaria.

108. The antibody, conjugate, or pharmaceutical composition for use according to any one of embodiments 102-107, wherein the method further comprises administering to the subject a second therapeutic agent.

109. An antibody, conjugate, or pharmaceutical composition for use according to embodiment 108, wherein the second therapeutic agent is a chemotherapeutic agent, a histone deacetylase inhibitor, an antibody, a cytokine, a tyrosine kinase inhibitor, an antihistamine, a leukotriene receptor antagonist, an immunomodulator, or an anti-inflammatory agent.

4. Description of the drawings

FIG. 1 shows full-length human KIT (SEQ ID NO: 1), genBank ^TM Amino acid sequence of accession number AAC 50969. First through fifth extracellular Ig-like domains (i.e., D1, D2, D3, D4, and D5) are shown; "{" shows the amino-terminus of each domainResidues and "}" show the carboxy-terminal residues of each domain. The D1 domain is shown at P34 to R112, the D2 domain is shown at D113 to P206, the D3 domain is shown at a207 to D309, the D4 domain is shown at K310 to N410, the hinge region between D4 and D5 is located at V409 to N410, and the D5 domain is shown at T411 to K509. The D1/D2 hinge region is located at D113 to L117; the D2/D3 hinge region is located between P206 and A210; and the D3/D4 hinge regions are located at D309 to G311. The D4/D5 region contains K310 through K509. The transmembrane domain comprises residues F525 to Q545 and the kinase domain comprises residues K589 to S933.

FIGS. 2A-2E show the effect of a particular anti-KIT antibody mAb1 on plasma tryptase levels according to the invention.

FIGS. 3A and 3B further illustrate the effect of a particular anti-KIT antibody mAb1 on plasma tryptase levels according to the invention.

FIG. 4 shows the effect of a particular anti-KIT antibody mAb1 on plasma Stem Cell Factor (SCF) levels according to the invention.

FIG. 5 shows the effect of a specific anti-KIT antibody mAb1 according to the invention and a corresponding antibody mAbc having the same variable region sequence but not mutated (wild-type) human IgG1 sequence on SCF-induced activation of wild-type KIT and downstream intracellular signaling pathways.

FIG. 6 shows the effect of a specific anti-KIT antibody mAb1 according to the invention and a corresponding antibody mAbc having the same variable domain sequence but the unmutated (wild-type) human IgG1 sequence on SCF-dependent cell proliferation.

FIG. 7 shows the binding affinities of a specific anti-KIT antibody mAb1 according to the invention and a corresponding antibody mAbc having the same variable domain sequence but the unmutated (wild-type) human IgG1 sequence to recombinant human Fc-gamma receptor (FcrR) and human neonatal Fc receptor (FcRn).

Figures 8A-8N show the binding curves of a specific anti-KIT antibody mAb1 according to the invention and a corresponding antibody mAbc having the same variable region sequence but not mutated (wild-type) human IgG1 sequence to recombinant human Fc-gamma receptor (FcrR) and human neonatal Fc receptor (FcRn). Fig. 8A shows the binding curve of mAb1 to fcri. Fig. 8B shows the binding curve of mAb1 to fcriia. Fig. 8C shows the binding curve of mAb1 to fcriib. Fig. 8D shows the binding curve of mAb1 to fcriiia. Fig. 8E shows the binding curve of mAb1 to fcriiib. Figure 8F shows the binding curve of mAb1 to FcRn (pH 6.0). Figure 8G shows the binding curve of mAb1 to FcRn (pH 7.2). Fig. 8H shows the binding curve of mAbc versus fcri. Fig. 8I shows the binding curve of mAbc to fcriia. FIG. 8J shows mAbc versus FcrRIIB binding curves. Fig. 8K shows the binding curve of mAbc to fcriiia. FIG. 8L shows mAbc versus FcrRIIIb binding curves. Figure 8M shows the binding curve of mAbc to FcRn (pH 6.0). Figure 8N shows the binding curve of mAbc to FcRn (pH 7.2).

FIG. 9 shows the effect of a particular anti-KIT antibody mAb1 according to the invention and a corresponding antibody mAbc having the same variable region sequence but an unmutated (wild-type) human IgG1 sequence on antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

FIG. 10 shows the effect of a particular anti-KIT antibody mAb1 on specific cytokine production according to the invention. The conditions shown for each histogram are from left to right: PHA, LPS, huIgG1 (soluble), mAb10.02nM (soluble), mAb 1.2 nM (soluble), mAb1 40nM (soluble), mAb 1.02 nM (dry-coated), mAb 1.2 nM (dry-coated) and mAb 1.40 nM (dry-coated).

Figure 11 shows a schematic showing the effect of KIT signal transduction in mast cells and the effect of mAb1 on KIT receptors.

Figures 12A-12D show that a single dose of mAb1 resulted in a rapid and sustained response, with a Complete Response (CR) rate of 95% in patients with chronic induced urticaria (CIndU). 10/10 cold urticaria (Cold U) patients achieved CR (FIG. 12A). 8/9 symptomatic skin Scarification (SD) patients achieved CR and 1/9SD patients achieved Partial Response (PR) (FIG. 12B). Cr=negative challenge test at ∈4 ℃ or 0 needle; pr=improvement by 4 ℃ or ≡2 needles; the maximum response per patient is shown. FIG. 12C shows time course in a ColdU patient As a result. In a complete cold patient (n=8), CR was maintained for a median duration of 77 days (fig. 12C) A. The invention relates to a method for producing a fibre-reinforced plastic composite FIG. 12D shows +.>As a result. In full SD patients (n=6), CR was maintained for a median duration of 57 days (fig. 12D).

FIGS. 13A-13B show overall disease improvement as demonstrated by physician global assessment (Phys-GA) and patient global assessment (Pat-GA). Phys-GA and Pat-GA were used to evaluate disease severity using the Likert scale of 0-3, where 0 is absent and 3 is severe.

Figures 14A-14D demonstrate that mAb1 treatment significantly depletes skin mast cells and serum tryptase. Fig. 14A shows mAb1 reduced skin mast cell number (n=14, p<0.05 represents p<0.01 x represents p<0.001, and p<0.0001). Fig. 14B shows mAb1 reduced serum tryptase below detection in all patients (the tryptase values below the assay limit (lloq=1 ng/mL) were normalized to 0). Figure 14C shows the kinetics of mast cells and tryptase. FIG. 14D shows that the number of skin mast cells correlates with serum tryptase levels (p<0.0001；R ² ＝0.45)。

Figures 15A-15D show that the kinetics of dermal mast cell and tryptase consumption reflect a decrease in the firing threshold. FIG. 15A shows mast cell kinetics and over time in ColdU patients As a result. FIG. 15B shows mast cell kinetics and +.>As a result. FIG. 15C shows tryptase kinetics and +.>Results (normalized to 0 for tryptase values below LLoQ; 3 ℃ for critical temperature threshold below 4 ℃ (negative test) were assigned). FIG. 15D shows tryptase kinetics and over time in SD patientsAs a result.

Figures 16A-16D show that the hematological parameters generally remained within normal ranges and a slight, transient and asymptomatic decrease in hemoglobin and White Blood Cell (WBC) parameters was noted. Fig. 16A shows hemoglobin (HgB) levels over time. Figure 16B shows WBC counts over time. Fig. 16C shows platelet counts over time. Fig. 16D shows Absolute Neutrophil Count (ANC) over time. In each figure, the hatched area indicates the corresponding normal range.

Figures 17A-17B show that a single 3mg/kg dose of mAb1 resulted in rapid and sustained improvement of urticaria control in cold urticaria (ColdU) patients (n=10, see figure 17A) and symptomatic skin Scars (SD) patients (n=10, see figure 17B). Urticaria Control Test (UCT) score=16 indicates complete control of urticaria, UCT score ∈12 indicates a well-controlled state of urticaria, and UCT score <12 indicates a poorly controlled state of urticaria. In each graph, the average UCT score ± SEM is shown.

FIGS. 18A-18B show that a single 3mg/kg dose of mAb1 results in rapid and sustained improvement in urticaria control in ColdU and SD patients. FIG. 18A shows that 100% of patients achieved a "well-controlled" state by week 8 (UCT score. Gtoreq.12). Fig. 18B shows that 63% of patients achieved a "full control" state by week 8 (UCT score=16).

Figures 19A-19B show that mAb1 greatly reduces the disease impact on quality of life for patients with cold urticaria (ColdU, n=10, see figure 19A) and symptomatic skin scars (SD, n=10, see figure 19B). Average DLQI score ± SEM are shown.

Figures 20A-20B show that mAb1 greatly reduces the disease impact on quality of life in cold urticaria (ColdU) and symptomatic skin Scars (SD) patients. Figure 20A shows that 93% of patients by week 4 achieved clinically significant quality of life improvement.The decrease in DLQI ≡4 is the least clinical weightDifference (MCID). * : only patients with a baseline DLQI score of > 4 were included. Figure 20B shows that 58% of patients reporting no disease impact on quality of life by week 4. />All responses provided weekly are included.

Figures 21A-21B show that a single 3mg/kg dose of mAb1 resulted in a rapid and durable improvement in challenge testing with 95% Complete Response (CR) and significant tryptase reduction. Figure 21A shows that mAb1 resulted in a rapid and durable improvement in the challenge test with a 95% complete response. By being used for cold urticaria (Cold U) Is based on the Critical Temperature Threshold (CTT) and on +.about.for symptomatic skin Scarification (SD)>Is used to evaluate disease activity. * : a value of 3 ℃ was assigned to the critical temperature threshold (negative test) below 4 ℃. By study, 10/10ColdU and 9/10SD patients underwent CR. Cr=negative challenge test at ∈4 ℃ (for cold) or 0 needle (for SD). In the figure, mean ± SEM are shown. Figure 21B shows mAb1 resulted in a rapid, durable and significant tryptase decrease. Trypsin-like values below the lower limit of quantitation (1 ng/mL) were normalized to 0. In the figure, mean ± SEM are shown.

5. Detailed description of the preferred embodiments

Provided herein are antibodies, and antigen-binding fragments thereof, that immunospecifically bind to human KIT (e.g., KIT polypeptides containing a human KIT D4 or D5 domain), and conjugates thereof. In a preferred embodiment, provided herein are antibodies that immunospecifically bind to human KIT, particularly antibodies having reduced Fc receptor binding activity (particularly reduced FcrR binding activity) and improved pharmacokinetics, reduced degranulation of FcgRI-expressing human mast cells, and/or reduced Fc receptor-dependent KIT agonist activity. In certain embodiments, provided herein are antigen binding fragments that immunospecifically bind to human KIT. Isolated nucleic acids (polynucleotides) encoding these antibodies or antigen binding fragments thereof are also provided. Vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding these antibodies or antigen binding fragments thereof are also provided. Methods of making these antibodies, cells, e.g., host cells, are also provided. Also provided herein are methods and uses for protecting, treating, or controlling a KIT-associated disorder or disease comprising administering an antibody or antigen-binding fragment thereof or conjugate thereof described herein. Also provided herein are methods of diagnosing a KIT-associated disorder or disease comprising contacting a sample with an antibody or antigen-binding fragment thereof described herein and determining the expression level of KIT in the sample relative to a reference sample (e.g., a control sample). Also provided herein are methods and uses of inhibiting the activity of KIT in a cell expressing KIT comprising contacting the cell with an effective amount of an antibody or antigen-binding fragment thereof described herein. Also provided herein are methods for inducing or enhancing cell differentiation or apoptosis in a cell expressing KIT comprising contacting the cell with an effective amount of an antibody or antigen-binding fragment thereof described herein.

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

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapy (e.g., an antibody or pharmaceutical composition provided herein) sufficient to reduce and/or ameliorate the severity and/or duration of a given disease and/or symptom associated therewith. These terms also encompass amounts necessary to reduce, slow or ameliorate the progression or development 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 effects of another therapy (e.g., a therapy other than an anti-KIT antibody provided herein). In some embodiments, an "effective amount" as used herein also refers to an amount of an antibody described herein that achieves the indicated result, e.g., a decrease in mast cell number and/or activity, a decrease in eosinophil number and/or activity, inhibition (e.g., partial inhibition) of KIT biological activity of a cell, such as inhibition of cell proliferation or cell survival, or enhancement or induction of apoptosis or cell differentiation, etc.

As used herein, the term "D4 or D5 region" or "D4/D5 domain" refers to the sequence from amino terminus to carboxy terminus: the D4, D4-D5 hinge region, and D5 cover regions within the KIT polypeptide of 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"). As used herein, amino acids V308 to H515 shown in fig. 1 are considered as examples of D4/D5 regions or domains.

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, an isoform of KIT, an interspecies homolog of KIT, or a KIT variant, e.g., a naturally occurring (e.g., allelic or splice variant, or mutant, e.g., somatic mutant) or an artificially constructed variant (e.g., recombinant or chemically modified variant). KIT is a type III receptor tyrosine kinase encoded by the c-KIT gene (see, e.g., yarden et al, nature,1986,323:226-232;Ullrich and Schlessinger,Cell,1990,61:203-212; clifford et al, j. Biol. Chem.,2003,278:31461-31464; yarden et al, EMBO j.,1987,6:3341-3351; mol et al, j. Biol. Chem.,2003,278: 31461-31464). GenBank ^TM Accession number NM 000222 provides an exemplary human KIT nucleic acid sequence. GenBank ^TM Exemplary human KIT amino acid sequences are provided by accession numbers NP 001087241, PI 0721 and AAC 50969. GenBank ^TM Accession number AAH75716 provides an exemplary murine KIT amino acid sequence. Natural KIT comprises 5 extracellular immunoglobulin (Ig) -like domains (D1, D2, D3, D4, D5), a single transmembrane region, an inhibitory cytoplasmic juxtamembrane junction separated by a kinase insertDomains and cytokinin kinase domains (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). FIG. 1 provides exemplary amino acid sequences of the D4/D5 region of human KIT at amino acid residues V308 through H515. In a specific embodiment, KIT is human KIT. In particular embodiments, KIT may exist as monomers, dimers, multimers, natural forms, or denatured forms.

As used herein, the term "combination" in the context of administration of other therapies refers to the use of more than one therapy. The use of the term "combination" does not limit the order in which the therapies are administered. The therapies may be administered, for example, sequentially, simultaneously or concomitantly.

As used herein, the terms "KIT-related disorder" or "KIT-related disease" are used interchangeably and refer to any disease that is caused, associated with, or caused, in whole or in part, by KIT expression and/or activity or lack thereof. In one aspect, the KIT-related disorder or disease may be known to or determinable by one of skill in the art. In certain embodiments, the KIT-associated disease or disorder is associated with KIT expression and/or activity. For example, KIT expression and/or activity may be combined with one or more other factors (e.g., mutation or expression and/or activity of another gene) to promote progression and/or development of a KIT-related disease or disorder. In certain embodiments, the KIT-associated disease or disorder is associated with one or more mutations in KIT.

In certain embodiments, the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In certain embodiments, the KIT-associated disorder is a fibrosis or inflammatory disorder, e.g., 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 particular embodiments, the KIT-related disorder is a mast cell-related disorder. In particular embodiments, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

As used herein, the terms "treatment" and "treating" refer to the reduction or improvement in development, severity, and/or duration of a KIT-associated disorder (e.g., cancer, inflammatory disorder, or fibrosis) caused by the administration of one or more therapies, including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as antibodies provided herein.

As used herein, the terms "control" and "management" refer to the beneficial effects of a subject obtained from therapy (e.g., a prophylactic or therapeutic agent) that do not result in a cure. In certain embodiments, one or more therapies (e.g., prophylactic or therapeutic agents, such as antibodies described herein) are administered to a subject to "control" a disorder or one or more symptoms thereof, thereby preventing the development or progression of the disorder.

As used herein, the term "protect" or "block" in the context of a disorder refers to 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 progress, recurrence, onset, or spread of the disorder and/or symptoms associated therewith caused by administration of a therapy or combination of therapies provided herein (e.g., a prophylactic or therapeutic agent, such as a combination of antibodies described herein).

As used herein, the term "prophylactic agent" refers to any agent that can completely or partially inhibit the progression, recurrence, onset, or spread of a disorder and/or symptoms associated therewith in a subject. In certain embodiments, the term "prophylactic agent" refers to an antibody described herein. In certain other embodiments, the term "prophylactic agent" refers to agents other than the antibodies described herein. In general, a prophylactic agent is an agent that is known to be useful for, or has been used for, or is being used for, preventing the onset, progression, development and/or severity of a disorder and/or a symptom associated therewith. In particular embodiments, the prophylactic agent is a human anti-KIT antibody, such as a humanized or fully human anti-KIT monoclonal antibody.

As used herein, the term "side effect" or "adverse effect" encompasses both an undesirable and adverse effect of a therapy (e.g., a prophylactic or therapeutic agent). The undesired effects are not necessarily disadvantageous. Adverse effects from therapy (e.g., prophylactic or therapeutic agents) can be detrimental or uncomfortable or dangerous. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, hair loss, asthma, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth and loss of appetite, rash or swelling at the site of administration, influenza-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Other undesirable effects experienced by patients are numerous and known in the art. Various effects are described in Physician's Desk Reference (71 st edition, 2017).

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, goat, rabbit, rat, mouse, etc.) or primate (e.g., monkey and human), e.g., human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a disorder. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a KIT-related disorder. In another embodiment, the subject is a non-human primate. In specific embodiments, the subject is an adult. In specific embodiments, the subject is an adult subject at least 18 years old. In particular embodiments, the subject is a child. In particular embodiments, the subject is a child between 1 year and 18 years of age. In particular embodiments, the subject is a human between 1 and 3 years of age. In particular embodiments, the subject is a human between 3 and 12 years of age or between 12 and 18 years of age.

As used herein, the term "therapy" may refer to any procedure, method, composition, formulation, and/or agent that may be used to prevent, protect, treat, control, or ameliorate a condition or disorder or symptom thereof, or one or more symptoms or conditions associated therewith. In certain embodiments, the term "therapy" refers to a drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful for protecting, treating, controlling, preventing, or ameliorating a condition or disorder or one or more symptoms thereof or one or more symptoms or conditions associated therewith. In certain embodiments, the term "therapy" refers to a therapy other than an anti-KIT antibody or pharmaceutical composition thereof described herein. In particular embodiments, "other therapies" and "additional therapies" refer to therapies other than treatment with an anti-KIT antibody or pharmaceutical composition thereof described herein. In particular embodiments, the therapy comprises the use of an anti-KIT antibody described herein as an adjuvant therapy. For example, the anti-KIT antibodies described herein are used in conjunction with drug therapies, biological therapies, surgery, and/or supportive therapies.

As used herein, the term "therapeutic agent" refers to any agent that can be used to protect, treat, control, or ameliorate a condition and/or symptoms associated therewith. In certain embodiments, the term "therapeutic agent" refers to an anti-KIT antibody or antigen-binding fragment thereof described herein. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody described herein. In particular embodiments, a therapeutic agent is an agent that is known to be useful for, or has been used or is currently being used for, the protection, treatment, control or amelioration of a disorder or one or more symptoms associated therewith.

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 terms "a" and "an" and "one or more" and "at least one" are used interchangeably herein.

It will be understood that wherever aspects are described herein by the language "comprising," similar aspects described by the terms "consisting of … …" and/or "consisting essentially of … …" are additionally provided.

As used herein and unless otherwise indicated, the terms "about" and "approximately" should be understood to allow standard changes, such as, for example, changes within 20% or 10% or 5%, at the discretion of the person skilled in the art. In particular embodiments, the terms "about" and "approximately" encompass the exact values recited.

5.1 antibodies

Provided herein are antibodies (e.g., anti-KIT antibodies) that specifically bind to a KIT receptor (e.g., the extracellular domain of a human KIT receptor, e.g., as set forth in SEQ ID NO:1 or fig. 1) or an antigen-binding fragment thereof.

As used herein, the terms "antibody" and "immunoglobulin" and "Ig" are terms of art and are used interchangeably herein and refer to a molecule having an antigen binding site that immunospecifically binds an antigen.

Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, and chimeric antibodies. In certain embodiments, the antibodies described herein represent a polyclonal antibody population. Antibodies may be immunoglobulin molecules of any type (e.g., igG, igE, igM, igD, igA or IgY), of any class (e.g., igG1, igG2, igG3, igG4, igA1 or IgA 2), or of any subclass (e.g., igG2a or IgG2 b). In certain embodiments, the antibodies described herein are IgG antibodies or classes (e.g., human IgG1 or IgG 4) or subclasses thereof.

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

As used herein, the terms "antigen binding domain," "antigen binding region," "antigen binding fragment," and similar terms refer to portions of an antibody molecule (e.g., complementarity Determining Regions (CDRs)) that comprise amino acid residues that interact with an antigen and confer specificity to the antigen on the antibody molecule. The antigen binding region may 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 by any method known to those skilled in the art. In particular, CDRs may be determined according to the Kabat numbering system (see Kabat et al (1991) Sequences of Proteins of Immunological Interest. (U.S. device of Health and Human Services, washington, D.C. 5 th edition). In certain aspects, may be according to (i) a Chothia numbering scheme, which will be referred to herein as a "Chothia CDR" (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. Pat. 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," in Antibody Engineering, kontermann and Dubel, chapter 31, pages 422-439, springer-Verlag, berlin (2001), or (iv) the Contact numbering system, which determines the CDR of antibodies based on analysis of the available complex crystal structure (bioinf. Org. Uk/abs) (see, e.g., macCallum et al, (1996) J Mol 5:732-745), in preferred embodiments, where the antigen binding antigen comprises a heavy IgG (e.g., a full length IgG, or a full length Fc region, such as a human IgG1, or a human Fc region, or a portion thereof, or a human Fc region, such as a 3, or a human Fc region or a 3 or a human fragment or 4.

As used herein, the term "constant region" or "constant domain" refers to a portion of an antibody that does not directly participate in binding of the antibody to an antigen, but that exhibits or contributes to a variety of effector functions, such as interactions with Fc receptors, e.g., the carboxy-terminal portion of the light and/or heavy chain. These terms refer to a portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.

As used herein, an "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. The region or polypeptide contributing to the epitope may be contiguous amino acids of the polypeptide or the epitope may be clustered together from two or more non-contiguous regions of the polypeptide.

When used in reference to antibodies, the term "heavy chain" as used herein refers to any of the different types based on the amino acid sequence of a constant domain, e.g., α (α), δ (δ), ε (ε), γ (γ), and μ (μ), which produce antibodies of IgA, igD, igE, igG and IgM classes, respectively, including subclasses of IgG, e.g., igG ₁ 、IgG ₂ 、IgG ₃ And IgG ₄ . In specific embodiments, the heavy chain is a human heavy chain.

As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex), as such binding is understood by those skilled in the art. For example, molecules that specifically bind to an antigen may bind to other peptides or polypeptides, typically with lower affinity, such as by, for example, immunoassays, biacore ^TM Determined by a KinExA 3000 instrument (Sapidyne Instruments, boise, ID) or other assay known in the art. In particular embodiments, molecules that immunospecifically bind to an antigen are used to bind K to another antigen than when the molecule is bound to another antigen _a K is at least 2log, 2.5log, 3log, 4log or more greater _a Binds to the antigen. In another embodiment, molecules that immunospecifically bind to an antigen do not cross-react with other proteins.In another embodiment, molecules that immunospecifically bind to an antigen do not cross-react with other non-KIT proteins.

As used herein, an "isolated" or "purified" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody was obtained, or substantially free of chemical precursors or other chemicals when chemically synthesized. In specific embodiments, an antibody or antigen binding fragment described herein is isolated.

The term "Kabat numbering" and similar terms are well-known in the art and refer to the numbering system of amino acid residues in the heavy and light chain variable regions of antibodies or antigen-binding portions thereof (Kabat et al (1971) Ann.NY Acad.Sci.190:382-391 and Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5 th edition, U.S. Pat. No. of Health and Human Services, NIH Publication No. 91-3242). CDRs within an antibody heavy chain molecule are typically found at amino acid positions 31 to 35 ("CDR 1"), amino acid positions 50 to 65 ("CDR 2") and amino acid positions 95 to 102 ("CDR 3") using the Kabat numbering system. CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR 1), amino acid positions 50 to 56 (CDR 2) and amino acid positions 89 to 97 (CDR 3) using the Kabat numbering system.

When used in reference to antibodies, the term "light chain" as used herein refers to any of a variety of types, e.g., kappa (kappa) and lambda (lambda) based on the amino acid sequence of a constant domain. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

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 will typically recognize a single epitope on the antigen. The term "monoclonal" is not limited to any particular method for producing antibodies. In general, a population of monoclonal antibodies can be produced by a cell, cell population, or cell line. In particular embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell (e.g., a host cell that produces a recombinant antibody), wherein the antibody immunospecifically binds to a KIT epitope (e.g., an epitope of D4 of human KIT), as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the examples provided herein. For example, monoclonal antibodies described herein can be prepared by hybridoma methods, such as Kohler et al; nature,256:495 (1975), or may be isolated from phage libraries, for example, using techniques as described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, e.g., chapter 11: short Protocols in Molecular Biology, (2002), 5 th edition, ausubel et al, main, john Wiley and Sons, new York). In particular embodiments, the monoclonal antibody is a monospecific antibody in which the antigen binding regions thereof are specific for the same epitope. In other embodiments, monoclonal monospecific antibodies may be monovalent (having one antigen binding region) or multivalent (having more than one antigen binding region), e.g., bivalent (having 2 antigen binding regions).

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

As used herein, the term "polyclonal antibody" refers to a population of antibodies that includes a plurality of different antibodies against the same or different epitopes within one or more antigens. Methods for producing polyclonal antibodies are known in the art (see, e.g., chapter 11: short Protocols in Molecular Biology, (2002), 5 th edition, ausubel et al, main, john Wiley and Sons, new York).

As used herein, the term "recombinant human antibody" includes human antibodies isolated, prepared, expressed or produced by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant, combinatorial human antibody libraries, antibodies isolated from animals (e.g., mice, rabbits, goats or cattle) transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., taylor, l.d. et al (1992) nucleic acids res.20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means, including, for example, by: synthesis, genetic engineering of DNA sequences encoding human immunoglobulin sequences, or sequences encoding human immunoglobulins, e.g., splicing of human immunoglobulin gene sequences to 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 have been modified, and as such the amino acid sequences of the VH and/or VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, are not naturally occurring within the human antibody germline repertoire in vivo. As a non-limiting example, a recombinant human antibody may be obtained by assembling some human sequence fragments into a composite human sequence of the recombinant human antibody.

As used herein, the term "variable region" or "variable domain" refers to an antibody portion, typically a light chain or heavy chain portion, typically from 110 to 120 amino acids at the amino-terminus in the mature heavy chain and from about 90 to 100 amino acids in the mature light chain, which varies widely in sequence among antibodies, and is used in the binding and specificity of a particular antibody for its particular antigen. The variability in the sequences is concentrated in those regions called Complementarity Determining Regions (CDRs), while the regions that are more highly conserved in the variable domains are called Framework Regions (FR).

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 particular embodiments, numbering of the amino acid positions of antibodies described herein is according to the EU index, e.g., kabat et al (1991) Sequences of Proteins of Immunological Interest, 5 th edition, U.S. Pat. No. of Health and Human Services, NIH Publication No.91-3242 ("Kabat et al"). In certain aspects, it may be according to (i) a Chothia numbering scheme, which will be referred to herein as a "Chothia CDR" (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. Pat. No.7,709,226); (ii) IMGT numbering systems, 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) ABM numbering systems, for example, 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," in Antibody Engineering, kontermann and Dubel Main, chapter 31, pages 422-439, springer-Verlag, berlin (2001); or (iv) a Contact numbering system that determines the CDRs of antibodies based on available complex crystal structure analysis (bioif. 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 a human Framework Region (FR). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) Framework Regions (FR). As a non-limiting example, the variable regions described herein result from assembling two or more fragments of a human sequence into a composite human sequence.

In a particular aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a light chain variable region ("VL") comprising VL CDRs 1-3 and a heavy chain variable region ("VH") comprising VH CDRs 1-3 as set forth in table 1. In a particular aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a light chain variable region ("VL") comprising VL CDRs 1-3 and a heavy chain variable region ("VH") comprising VH CDRs 1-3 as set forth in table 2 (group 1 or group 2). In a particular aspect, an anti-KIT antibody (e.g., a humanized antibody) provided herein comprises a light chain variable region ("VL") comprising VL CDRs 1-3 and a heavy chain variable region ("VH") comprising VH CDRs 1-3 (AbM CDRs or Contact CDRs) as set forth in table 3.

In a particular aspect, an anti-KIT antibody (e.g., humanized antibody) provided herein comprises a VL comprising VL CDRs 1-3 (SEQ ID NOS: 2-4) as shown in Table 1 and a VH comprising VHCDR 1-3 (SEQ ID NOS: 5-7) as shown in Table 1. In particular embodiments, such anti-KIT antibodies are naked antibodies. In particular embodiments, such anti-KIT antibodies are bivalent monospecific antibodies. In particular embodiments, such anti-KIT antibodies are dual specificity antibodies. In certain embodiments, such anti-KIT antibodies are not dual specific antibodies.

Table 1: CDR amino acid sequences

	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

Table 2: CDR amino acid sequences

Table 3: CDR amino acid sequences

In a particular aspect, an anti-KIT antibody (e.g., humanized antibody) provided herein comprises:

(i) VL comprising an 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

(ii) VH comprising the 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 Is any amino acid.

In a specific embodiment, xκ ₁ Is an amino acid having an aromatic or aliphatic hydroxyl side chain, xκ ₂ Is an amino acid having an aliphatic or aliphatic hydroxyl side chain, xκ ₃ Is an amino acid having 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, X _K6 Is an amino acid having an aromatic side chain, X _H1 Is an amino acid having an aliphatic side chain, X _H2 Is an amino acid having an aliphatic side chain, X _H3 Is an amino acid having a polar or basic side chain, X _H4 Is an amino acid having an aliphatic side chain, X _H5 Is an amino acid having an aliphatic side chain, X _H6 Is an amino acid with an acidic side chain, X _H7 Is an amino acid having acidic and amide derivative side chains, and X _H8 Is an amino acid having an aliphatic hydroxyl side chain.

In a specific embodiment, X _K1 Is amino acid F or S, xkappa ₂ Is amino acid A or S, X _K3 Is the amino acid T or S, X _K4 Is the 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 the 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 Is amino acid S or T.

(i) VL comprising an amino acid sequence:

(ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 comprising the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7.

In a specific embodiment, xκ ₁ Is an amino acid having an aromatic or aliphatic hydroxyl side chain, xκ ₂ Is an amino acid having an aliphatic or aliphatic hydroxyl side chain, xκ ₃ Is an amino acid having 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 having a charged or acidic side chain and X _K6 Is an amino acid having an aromatic side chain.

In a specific embodiment, X _K1 Is amino acid F or S, xkappa ₂ Is amino acid A or S, X _K3 Is the amino acid T or S, X _K4 Is the amino acid S or P, X _K5 Is amino acid D or T and X _K6 Is amino acid F or Y.

(i) A VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide having an amino acid sequence shown in FIG; and

(ii) VH comprising the amino acid sequence:

QVQLVQSGAEX _H1 KKPGASVKX _H2 SCKASGYTFTDYYINWVX _H3 QAPGKGLEWIA RIYPGSGNTYYNEKFKGRX _H4 TX _H5 TAX _H6 KSTSTAYMX _H7 LSSLRSEDX _H8 AVYFCARG VYYFDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X _H1 To X _H8 Is any amino acid.

In a specific embodiment, X _H1 Is an amino acid having an aliphatic side chain, X _H2 Is an amino acid having an aliphatic side chain, X _H3 Is an amino acid having a polar or basic side chain, X _H4 Is an amino acid having an aliphatic side chain, X _H5 Is an amino acid having an aliphatic side chain, X _H6 Is an amino acid with an acidic side chain, X _H7 Is an amino acid having acidic and amide derivative side chains, and X _H8 Is an amino acid having an aliphatic hydroxyl side chain.

In a specific embodiment, 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 the 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 Is amino acid S or T.

In particular aspects, anti-KIT antibodies (e.g., humanized antibodies) provided herein comprise 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 particular embodiments, such anti-KIT antibodies are naked antibodies. In particular embodiments, such anti-KIT antibodies are bivalent monospecific antibodies. In particular embodiments, such anti-KIT antibodies are dual specificity antibodies. In certain embodiments, such anti-KIT antibodies are not dual specific antibodies.

Table 4: VH amino acid sequence

Table 5: VL amino acid sequence

In a particular aspect, an anti-KIT antibody (e.g., humanized antibody) provided herein comprises: comprising SEQ ID NO:8, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:8, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:14, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:8, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:15, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:8, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:16, and a VL of the amino acid sequence shown in seq id no.

In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:9, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:9, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:14, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:9, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:15, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:9, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:16, and a VL of the amino acid sequence shown in seq id no.

In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:10, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:10, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:14, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:10, and a VH comprising the amino acid sequence set forth in SEQ ID NO:14, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:10, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:15, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:10, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:16, and a VL of the amino acid sequence shown in seq id no.

In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:11, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:11, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:14, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:11, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:15, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:11, and/or a VH comprising the amino acid sequence set forth in SEQ ID NO:16, and a VL of the amino acid sequence shown in seq id no.

In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:12, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:12, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:14, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:12, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:15, and a VL of the amino acid sequence shown in seq id no. In one embodiment, an anti-KIT antibody provided herein comprises: comprising SEQ ID NO:12, and/or VH comprising the amino acid sequence set forth in SEQ ID NO:16, and a VL of the amino acid sequence shown in seq id no.

(i) VL comprising the amino acid sequence: and SEQ ID NO:13 has at least 90% identity to SEQ ID NO:14 has at least 88% identity to SEQ ID NO:15, or at least 87% identical to SEQ ID NO:16 having an amino acid sequence with at least 84% identity; and

(ii) VH comprising the amino acid sequence: and SEQ ID NO:8 has at least 93% identity to SEQ ID NO:9 has at least 92% identity to SEQ ID NO:10 has at least 90% identity to SEQ ID NO:11 or at least 87% identical to SEQ ID NO:12, an amino acid sequence having at least 86% identity.

Previous anti-KIT antibodies have been found to cause degranulation of FcgRI expressing human mast cells and/or exhibit Fc receptor-dependent KIT agonist activity, which can lead to undesired infusion-related responses (IRR) as well as other adverse effects.

In various embodiments, an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, 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 (specifically reduced FcrR binding activity), do not cause degranulation of FcgRI-expressing human mast cells 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 caused by a modified (e.g., mutated) Fc region or domain.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein has reduced Fc receptor binding activity (specifically reduced FcrR binding activity). In particular embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not have significant Fc receptor (particularly FcrR) binding activity. In particular embodiments, the anti-KIT antibodies or antigen-binding fragments described herein have no detectable Fc receptor (specifically FcrR) binding activity. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein has 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% lower Fc receptor (specifically FcrR) binding activity than an appropriate control antibody or antigen-binding fragment. When an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (e.g., mutated) human IgG1, igG2, igG3, or IgG4 Fc region or domain, in preferred embodiments, a suitable 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 particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein does not cause significant degranulation of human mast cells expressing FcgRI (e.g., as determined, for example, by the percentage of β -hexosaminidase released from human mast cells in culture (e.g., in the presence of ifnγ). In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein does not cause detectable degranulation of human mast cells expressing FcgRI (e.g., as determined, for example, by the percentage of β -hexosaminidase released from human mast cells in culture (e.g., in the presence of ifnγ). In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein results in 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% lower degranulation of human mast cells expressing FcgRI (e.g., as determined, for example, by the percentage of β -hexosaminidase released from human mast cells in culture (e.g., in the presence of ifny) compared to an appropriate control antibody or antigen-binding fragment. In particular embodiments, the release of β -hexosaminidase in human mast cells in culture is reduced by more than 50% by an anti-KIT antibody or antigen-binding fragment described herein in the presence of ifnγ as compared to an appropriate control antibody or antigen-binding fragment. In particular embodiments, the release of β -hexosaminidase in human mast cells in culture is reduced by more than 60%, more than 70%, or more than 80% by an anti-KIT antibody or antigen-binding fragment described herein in the presence of ifnγ as compared to an appropriate control antibody or antigen-binding fragment. When an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (e.g., mutated) human IgG1, igG2, igG3, or IgG4 Fc region or domain), in a preferred embodiment, a suitable control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL, but having the same isotype of wild-type (unmodified) Fc region or domain. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) human IgG1Fc region or domain and results in 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% lower degranulation of human mast cells expressing FcgRI (e.g., as determined, for example, by the percentage of β -hexosaminidase released from human mast cells in culture (e.g., in the presence of ifnγ) as compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but having a wild-type (unmodified) human IgG1Fc region or domain. In particular embodiments, the release of β -hexosaminidase from human mast cells in culture is reduced by more than 50% in the presence of ifnγ by an anti-KIT antibody or antigen-binding fragment described herein comprising a modified (e.g., mutated) human IgG1Fc region or domain, as compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but having a wild-type (unmodified) human IgG1Fc region or domain. In particular embodiments, the release of β -hexosaminidase from human mast cells in culture is reduced by more than 60%, more than 70%, or more than 80% in the presence of ifny by an anti-KIT antibody or antigen-binding fragment described herein comprising a modified (e.g., mutated) human IgG1Fc region or domain, as compared to a corresponding antibody or antigen-binding fragment having the same VH and VL but having a wild-type (unmodified) human IgG1Fc region or domain.

In particular embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not exhibit significant Fc receptor-dependent KIT agonistic activity (e.g., as determined, for example, by KIT phosphorylation). In particular embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not exhibit detectable Fc receptor-dependent KIT agonistic activity (e.g., as determined, for example, by KIT phosphorylation). In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein results in 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% lower Fc receptor-dependent KIT activity (e.g., as determined, for example, by KIT phosphorylation) than an appropriate control antibody or antigen-binding fragment. In particular embodiments, the Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation using a cross-linked Fc receptor) is reduced by more than 50% using an anti-KIT antibody or antigen-binding fragment described herein as compared to an appropriate control antibody or antigen-binding fragment. In particular embodiments, the Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation using a cross-linked Fc receptor) is reduced by more than 60%, more than 70%, or more than 80% using an anti-KIT antibody or antigen-binding fragment described herein as compared to an appropriate control antibody or antigen-binding fragment. When an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) Fc region or domain (e.g., a modified (e.g., mutated) human IgG Fc region or domain, such as a modified (e.g., mutated) human IgG1, igG2, igG3, or IgG4 Fc region or domain), in a preferred embodiment, a suitable control antibody or antigen-binding fragment is an antibody or antigen-binding fragment having the same VH and VL, but having the same isotype of wild-type (unmodified) Fc region or domain. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein comprises a modified (e.g., mutated) human IgG1 Fc region or domain and results in 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% lower Fc receptor-dependent KIT activity (e.g., as determined, for example, by KIT phosphorylation) when compared to 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 particular embodiments, the anti-KIT antibodies or antigen-binding fragments described herein do not exhibit significant or detectable Fc receptor-dependent KIT agonistic activity as described herein, even when crosslinked on THP-1 cells. In particular embodiments, using an anti-KIT antibody or antigen-binding fragment comprising a modified (e.g., mutated) human IgG1 Fc region or domain as described herein, the Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation using a cross-linked Fc receptor) is reduced by more than 50% when compared to 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 particular embodiments, using an anti-KIT antibody or antigen-binding fragment comprising a modified (e.g., mutated) human IgG1 Fc region or domain as described herein, the Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation using a cross-linked Fc receptor) is reduced by more than 60%, more than 70%, or more than 80% when compared to 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 various embodiments, an anti-KIT antibody or antigen binding fragment described herein (1) reduces disease activity in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (2) reduces the number of skin mast cells in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (3) reduces tryptase levels in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (4) improves urticaria control in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), (5) improves quality of life in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment), and/or (6) maintains a blood plasma parameter (e.g., a white blood cell count (hc) in a blood cell count (hc) or normal blood cell count (hc) in a range of a patient, e.g., a WBC.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein can significantly reduce the risk of a patient with CIndU (e.g., a patient with CIndU that is refractory to antihistamine treatment) in a patient with CIndU relative to a pre-treatment value Is included in the temperature range. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein can treat a patient with CIndU (e.g., a patient with CIndU that is refractory to antihistamine treatment) at ∈>At least 5 ℃, at least 6 ℃, at least 7 ℃, at least 8 ℃, at least 9 ℃, at least 10 ℃, at least 11 ℃, at least 12 ℃, at least 13 ℃, at least 14 ℃, at least 15 ℃, at least 16 ℃, at least 17 ℃, at least 18 ℃, at least 19 ℃, or at least 20 ℃ (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein can significantly reduce the number of patients with CIndU (e.g., CIndU patients whose CIndU is refractory to antihistamine treatment) in relation to the needle count prior to treatmentThe number of needles in the needle bed. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein can treat a patient with CIndU (e.g., a patient with CIndU that is refractory to antihistamine treatment) in relation to the needle count prior to treatment At least 1, at least 2, at least 3, or at least 4 (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein may significantly improve physician global assessment (Phys-GA) and/or patient global assessment (Pat-GA) relative to pre-treatment levels in particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein may improve physician global assessment (Phys-GA) and/or patient global assessment (Pat-GA) relative to pre-treatment levels by reducing the Likert scale (0-3, where 0 is absent and 3 is severe) by at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, or at least 1.3 (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment). 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein can significantly reduce the number of dermal mast cells in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) relative to the number prior to treatment. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein can reduce the number of skin mast cells in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by at least 20%, at least 40%, at least 60%, or at least 80% (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) relative to the number prior to treatment. 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein can significantly reduce serum tryptase in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) relative to a pre-treatment level. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein can reduce serum tryptase in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by at least 50%, at least 70%, or at least 90% (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment) relative to a pre-treatment level. 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein can significantly improve urticaria control in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) relative to the pre-treatment levels. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein can improve urticaria control in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by increasing the Urticaria Control Test (UCT) score by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16, or by increasing the UCT score by at least 12, at least 13, at least 14, at least 15, or 16, relative to the pre-treatment level (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein can significantly improve quality of life in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) relative to a pre-treatment level. In particular embodiments, an anti-KIT antibody or antigen-binding fragment described herein can improve quality of life in a CIndU patient (e.g., a CIndU patient whose CIndU is refractory to antihistamine treatment) by reducing the dermatological quality of life index (DLQI) to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or at least 25, or by reducing DLQI by at most 5, at most 4, at most 3, at most 2, at most 1, or 0, relative to a pre-treatment level (e.g., within 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks after treatment with the anti-KIT antibody or antigen-binding fragment). In 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.

In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein maintains a hematological parameter in a patient, such as hemoglobin (HgB) level, white Blood Cell (WBC) count, platelet count, and/or Absolute Neutrophil Count (ANC), within a normal range. In certain embodiments, an anti-KIT antibody or antigen-binding fragment described herein maintains hematological parameters, such as hemoglobin (HgB) levels, white Blood Cell (WBC) counts, platelet counts, and/or Absolute Neutrophil Counts (ANC), in a patient with CIndU (e.g., a patient with CIndU who is refractory to antihistamine treatment). In specific embodiments, the hematology parameter is 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.

In various embodiments, an anti-KIT antibody described herein has one or more of the properties described herein. In various embodiments, the antigen-binding fragments of the anti-KIT antibodies described herein have one or more of the properties described herein.

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

In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG1 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue selected from the group consisting of: as numbered by EU numbering as described in Kabat, 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, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, Y, 263E, 265G, 263N, Q, Y, 265F, 265V, 265H, 265V, 266A, 61T, 61H, 61T. 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 322Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332N, 332T, 332H, 332Y and 332H. Optionally, the Fc region or domain may include other and/or alternative non-naturally occurring amino acid residues known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;6,277,375;6,737,056; PCT patent publication WO 01/58957; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217). In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG2 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to an amino acid residue described herein for a human IgG1Fc region or domain, as can be determined by one of skill in the art. In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG3 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to an amino acid residue described herein for a human IgG1Fc region or domain, as can be determined by one of skill in the art. In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG4 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to an amino acid residue described herein for a human IgG1Fc region or domain, as can be determined by one of skill in the art.

In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG1 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue selected from the group consisting of amino acid residues numbered as set forth by the EU numbering described in Kabat, 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, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, Y, 263E, 265G, 263N, Q, Y, 265F, 265V, 265H, 265V, 266A, 61T, 61H, 61T. 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 322Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332N, 332T, 332H, 332Y and 332H. . Optionally, the Fc region or domain may include other and/or alternative non-naturally occurring amino acid residues known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;6,277,375;6,737,056; PCT patent publication WO 01/58957; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217). In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG2 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to an amino acid residue described herein for a human IgG1Fc region or domain, as can be determined by one of skill in the art. In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG3 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to an amino acid residue described herein for a human IgG1Fc region or domain, as can be determined by one of skill in the art. In particular embodiments, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of human IgG4 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue that is equivalent to an amino acid residue described herein for a human IgG1Fc region or domain, as can be determined by one of skill in the art.

In a certain aspect, provided herein is an antibody comprising an Fc region or domain, wherein said Fc region or domain is an Fc region or domain of human IgG1 and comprises at least a non-naturally occurring amino acid at one or more positions selected from 239, 330 and 332, as numbered by EU numbering as set forth in Kabat. In particular embodiments, provided herein are antibodies 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 239D, 330L, and 332E, as numbered by EU numbering as set forth in Kabat. Optionally, the Fc region or domain may also include other non-naturally occurring amino acids at one or more positions selected from 252, 254, and 256, as numbered by EU numbering as set forth in Kabat. In particular embodiments, provided herein are antibodies 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 239D, 330L, and 332E, as numbered by EU numbering as set forth in Kabat, and the at least one non-naturally occurring amino acid located at one or more positions is selected from 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat. In particular embodiments, provided herein are antibodies comprising an Fc region or domain, wherein the Fc region or domain is that of a human IgG2, igG3, or IgG4 and comprises at least one non-naturally occurring amino acid residue equivalent to the amino acid residues described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In particular embodiments, provided herein are antibodies 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 at one or more positions equivalent to those described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In one embodiment, the Fc region or domain comprising such sequences exhibits one or more Fc activities, e.g., binding affinity for Fc receptors or effector functions, such as ADCC or CDC. In particular embodiments, the Fc region or domain comprising such a sequence exhibits reduced Fc activity, e.g., reduced binding affinity to Fc receptors or reduced effector functions, such as ADCC or CDC. In particular embodiments, the Fc region or domain comprising such a sequence exhibits enhanced FcRn activity, e.g., enhanced half-life.

Other 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 U S A92:11980-11984; 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 Immunol157:4963-4969; armour et al 1999,Eur J Immunol 29:2613-2624; idusogie et al, 2000,JImmunol 164:4178-4184; reddy et al, 2000,J Immunol 164:1925-1933; xu et al, 2000,Cell Immunol 200:16-26; idusogie et al, 2001,J Immunol 166:2571-2575; shields et al 2001,J Biol Chem 276:6591-6604; jefferis et al, 2002,Immunol Lett 82:57-65; presta et al, 2002,Biochem Soc Trans 30:487-490; U.S. Pat. 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;6,821,505;6,277,375;8,163,882;7,355,008;7,960,512;8,039,592;8,039,359;8,101,720;7,214,775;7,682,610;7,741,442; U.S. patent publication No.2004/0002587 and PCT patent publication WO 94/29351; WO 99/58372; WO 00/42072; WO 04/029207; WO 04/099249; WO 04/063151.

In particular embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG1Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by EU numbering as set forth in Kabat. In particular embodiments, the modified (e.g., mutated) human IgG1Fc region or domain further comprises non-naturally occurring amino acids 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat.

In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG2Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, and 322Q numbered as by EU numbering as set forth for the human IgG1Fc region or domain in Kabat, as can be determined by one of skill in the art. In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG2Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E numbered as described by EU numbering for human IgG1Fc regions or domains in Kabat, as can be determined by one of skill in the art.

In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG3Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, and 322Q numbered as by EU numbering as set forth for the human IgG1 Fc region or domain in Kabat, as can be determined by one of skill in the art. In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG3Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E numbered as described by EU numbering for human IgG1 Fc regions or domains in Kabat, as can be determined by one of skill in the art.

In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG4Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, and 322Q numbered as by EU numbering as set forth for the human IgG1 Fc region or domain in Kabat, as can be determined by one of skill in the art. In certain embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG4Fc region or domain comprising non-naturally occurring amino acids equivalent to 234A, 235Q, 322Q, 252Y, 254T, and 256E numbered as described by EU numbering for human IgG1 Fc regions or domains in Kabat, as can be determined by one of skill in the art.

In particular embodiments, the antibodies described herein comprise VL and VH CDR sequences described 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 non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by EU numbering as described in Kabat.

In preferred embodiments, the antibodies described herein comprise VL and VH CDR sequences described 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 non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by EU numbering as described in Kabat.

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

(i) A VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide having an amino acid sequence shown in FIG; (ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by EU numbering as set forth in Kabat.

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

(i) A VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 having the amino acid sequences of SEQ ID NOs: 2. SEQ ID NO:3 and SEQ ID NO:4, and a polypeptide having an amino acid sequence shown in FIG; (ii) A VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, the VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 5. SEQ ID NO:6 and SEQ ID NO: 7; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat.

In another aspect, provided herein is an antibody that immunospecifically binds to human KIT comprising: (i) VL comprising the amino acid sequence: DIVMTQSPSX _K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX _K2 LIYSASYR YSGVPDRFX _K3 GSGSGTDFTLTISSLQX _K4 EDFAX _K5 YX _K6 CQQYNSYPRTFGGGTKVEIK (SEQ ID NO: 17), wherein X _K1 Is an amino acid having an aromatic or aliphatic hydroxyl side chain, X _K2 Is an amino acid having an aliphatic or aliphatic hydroxyl side chain, X _K3 Is an amino acid having 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 having a charged or acidic side chain and X _K6 Is an amino acid having an aromatic side chain; and (ii) VH comprising the amino acid sequence: QVQLVQSGAEX _H1 KKPGASVKX _H2 SCKASGYTFTDYYINWVX _H3 QAPGKGLEWIARIY PGSGNTYYNEKFKGRX _H4 TX _H5 TAX _H6 KSTSTAYMX _H7 LSSLRSEDX _H8 AVYFCARGVYY FDYWGQGTTVTVSS (SEQ ID NO: 18), wherein X _H1 Is provided withAmino acids of aliphatic side chains, X _H2 Is an amino acid having an aliphatic side chain, X _H3 Is an amino acid having a polar or basic side chain, X _H4 Is an amino acid having an aliphatic side chain, X _H5 Is an amino acid having an aliphatic side chain, X _H6 Is an amino acid with an acidic side chain, X _H7 Is an amino acid having acidic and amide derivative side chains, and X _H8 Is an amino acid having an aliphatic hydroxyl side chain; and (iii) a modified (e.g., mutated) human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, 322Q, and preferably further comprising 252Y, 254T, and 256E, as numbered by EU numbering as set forth in Kabat.

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

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

In particular embodiments, the antibodies provided herein comprise: a heavy chain comprising the amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYINWVRQAPGKGLEWIARIYPGSGNTYYNEKFKGRATLTADKSTSTAYMQLSSLRSEDTAVYFCARGVYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21).

In particular embodiments, the antibodies provided herein comprise: a light chain comprising the amino acid sequence: DIVMTQSPSSLSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

In particular embodiments, the antibodies provided herein comprise: a heavy chain comprising the amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTDYYINWVRQAPGKGLEWIARIYPGSGNTYYNEKFKGRATLTADKSTSTAYMQLSSLRSEDTAVYFCARGVYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAQGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 21); and a light chain comprising the amino acid sequence: DIVMTQSPSSLSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 22).

In particular embodiments, provided herein are antibodies comprising: (i) a heavy chain comprising the 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, mutations in the constant region are shown in double underline (as compared to wild type human IgG 1); and

(ii) A light chain comprising the amino acid sequence:

wherein the leader sequence is shown in bold italics, the variable region (VL) is shown in italics and the constant region is shown underlined.

In particular embodiments, an anti-KIT antibody described herein does not bind to (e.g., has undetectable binding to) any human Fc-gamma receptor (FcrR receptor). In particular embodiments, the anti-KIT antibodies described herein do not bind to (e.g., have undetectable binding to) human fcri. In particular embodiments, an anti-KIT antibody described herein does not bind to (e.g., has undetectable binding to) human fcriia. In particular embodiments, an anti-KIT antibody described herein does not bind to (e.g., has undetectable binding to) human fcriib. In particular embodiments, the anti-KIT antibodies described herein do not bind to (e.g., have undetectable binding to) human fcriiia. In particular embodiments, an anti-KIT antibody described herein does not bind to (e.g., has undetectable binding to) human fcriiib.

In particular 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 10000-fold higher binding affinity) to 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 particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 20nM at pH 6.0. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 2nM at pH 6.0. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 1nM at pH 6.0. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 500nM at pH 6.0. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 400pM at pH 6.0. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 200nM at pH 7.2. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 150nM at pH 7.2. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 100nM at pH 7.2. In particular embodiments, an anti-KIT antibody described herein binds to FcRn with a KD of less than 80nM at pH 7.2.

In particular 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 antibody-independent cell-mediated cytotoxicity (ADCC). In particular 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 (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) ADCC relative to a corresponding antibody having the same variable region sequence but having an unmodified (wild-type) human IgG constant region.

In particular 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 (e.g., at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or at least 99% less) cytokine production (e.g., IFN- γ, IL-1β, IL-2, IL-6, IL-8, IL-10, and/or TNF- α) relative to a corresponding antibody having the same variable region sequence but having an unmodified (wild-type) human IgG constant region.

In a particular aspect, also provided are antigen-binding fragments of the antibodies described herein, preferably antigen-binding fragments comprising 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). In a particular aspect, also provided are antigen binding fragments of the antibodies described herein, comprising a portion of a heavy chain Fc region or domain (e.g., a portion of a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region or domain).

In certain aspects, anti-KIT antibodies or antigen-binding fragments thereof may be obtained using methods known in the art, e.g., see section 5.4 below.

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

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to KIT, e.g., the D4 or D4/D5 region of human KIT, and has at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher affinity for KIT antigen consisting essentially of only the D4 domain than KIT antigen consisting essentially of only the D5 domain.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to a KIT polypeptide(e.g., D4 region of human KIT), its EC ₅₀ The (half maximal effect concentration) value is about 50nM, 10nM, 500pM, 300pM, 200pM, 100pM, or 50pM or less, as determined by assays described in the art, such as ELISA.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to a KIT polypeptide (e.g., the D4 region of human KIT), the EC thereof ₅₀ Values of about 200pM or 150pM or less, as determined by assays described in the art, such as ELISA or FACs using CHO-WT-KIT cells (CHO cells engineered to recombinantly express wild-type human KIT).

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of an IC of about 600pM or less ₅₀ (50% inhibition concentration) values block KIT phosphorylation.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of internalizing or enhancing KIT receptor by, e.g., 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 unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art. In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of internalizing a KIT receptor by, e.g., at least about 25% or 35%, optionally to about 75%, relative to internalization in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art. In particular embodiments, an anti-KIT antibody provided herein, or antigen-binding fragment thereof, is capable of internalizing or enhancing 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 internalization in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art. Techniques for quantification or visualization of cell surface receptors are well known in the art and include a variety of fluorescent and radioactive techniques. For example, one method involves incubating the cells with a radiolabeled anti-receptor antibody. Alternatively, the natural ligand of the receptor may be conjugated to a fluorescent molecule or radiolabel and incubated with the cell. Other receptor internalization assays are well known in the art and are described, for example, in Jisenez 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.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of causing 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 turnover in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art (e.g., pulse-chase assays). In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of eliciting or enhancing KIT receptor turnover by at least about 25% or 35%, optionally to about 75%, relative to turnover in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art (e.g., pulse-chase assays). In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of causing 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 relative to turnover in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT) A factor of 30, 40, 50, 60, 70, 80, 90, or 100 as assessed by methods described herein or known to those of skill in the art (e.g., pulse-chase assays). Methods for determining receptor turnover are well known in the art. For example, one can use ³⁵ S-EXPRESS protein labeling mixtures (NEG 772, NEN Life Science Products) pulse labeled KIT-expressing cells, wash and track with unlabeled vehicle for a period of time, then immunoprecipitate protein lysates from the labeled cells using anti-KIT antibodies, and separate and visualize by SDS-PAGE (e.g., exposure to PhosphoImager screen (Molecular Dynamics), scan using Typhoon8600 scanner (Amersham) and analyze using ImageQuant software (Molecular Dynamics) (see, e.g., chan et al, development,2004, 131:5551-5560).

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of causing or enhancing KIT receptor degradation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to degradation in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art (e.g., pulse-chase assays). In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of causing or enhancing KIT receptor degradation by at least about 25% or 35%, optionally to about 75%, relative to degradation in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT), as assessed by methods described herein or known to those of skill in the art (e.g., pulse-chase assays). In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is capable of causing or enhancing KIT receptor degradation by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold relative to degradation in the presence of an unrelated antibody (e.g., an antibody that does not immunospecifically bind to KIT) A factor of 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 as assessed by methods described herein or known to those of skill in the art (e.g., pulse-chase assays). Techniques for determining or monitoring ubiquitination and/or degradation (e.g., kinetics or degradation rate) of cell surface receptors are well known in the art and include a variety of fluorescence and radioactivity techniques (see, e.g., international patent application publication No. wo 2008/153926 A2). For example, the use of radiolabeled ligands, such as ¹²⁵ One or more pulse-chase experiments of I-SCF to quantitatively measure the degradation of KIT.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein does not bind to an extracellular ligand binding site of KIT, e.g., an SCF binding site of KIT. In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein does not inhibit ligand binding to KIT, e.g., does not inhibit KIT ligand (e.g., SCF) binding to KIT, as determined by methods described in the art, e.g., ELISA. In certain embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein does not completely inhibit or partially inhibit binding to a ligand of KIT, e.g., does not completely inhibit or partially inhibit binding to a KIT ligand of KIT (e.g., SCF), as determined by methods described in the art, e.g., ELISA or FACS (flow cytometry fluorescence sorting technique).

In particular aspects, an anti-KIT antibody (e.g., a human or humanized antibody) provided herein is an inhibitory antibody, i.e., an antibody that inhibits (e.g., partially inhibits) KIT activity, i.e., one or more KIT activities. In particular embodiments, partial inhibition of KIT activity results in, for example, about 25% to about 65% or 75% inhibition. In particular embodiments, 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 particular embodiments, an antibody described herein inhibits KIT phosphorylation (e.g., ligand-induced phosphorylation).

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT tyrosine phosphorylation in a KIT cytoplasmic domain.

In another embodiment, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits cell proliferation, e.g., mast cell proliferation or eosinophil proliferation. In another embodiment, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits cell survival, e.g., 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%.

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

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits 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, eosinophil or mast cell degranulation is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

In another embodiment, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits mast cell vehicle release. In certain aspects, the mast cell vehicle release is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Assays have been described that measure mast cell activity, such as release of mediators from mast cell cultures, such as rodent and human mast cell cultures (see, e.g., kuehn et al, "Measuring Mast Cell Mediator Release," in Current Protocols in Immunology, unite7.38.1-7.38.9,2010, month 11 (John Wiley & Sons, inc.). 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.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein induces apoptosis, e.g., mast cell apoptosis or eosinophil apoptosis. In another embodiment, an anti-KIT antibody or antigen-binding fragment thereof provided herein induces cell differentiation, e.g., mast cell differentiation.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein may achieve any one of the following: a decrease in eosinophil number and/or activity, a decrease in mast cell proliferation, a decrease in plasma tryptase levels, a decrease in plasma SCF levels, a decrease in mast cell number or amount, an inhibition or decrease in mast cell activity, a decrease in mast cell-induced inflammatory factor production or release, a decrease in inflammatory factor release, a restoration of mast cell homeostasis, a decrease in mast cell migration, a decrease in mast cell adhesion, an inhibition or decrease in mast cell recruitment by eosinophils, and an inhibition or decrease in antigen-mediated degranulation of mast cells.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT activity but does not inhibit KIT dimerization. In another embodiment, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT activity and does not inhibit ligand binding to KIT, e.g., does not inhibit KIT ligand (e.g., SCF) binding to KIT, but does inhibit KIT dimerization.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT activity, such as ligand-induced tyrosine phosphorylation of KIT cytoplasmic domains, by about 25% to about 65% or 75% as determined by cell-based phosphorylation assays well known in the art, e.g., cell-based phosphorylation assays described herein. In certain embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits KIT activity, such as ligand-induced tyrosine phosphorylation of a KIT cytoplasmic domain, by about 35% to about 85% or 95%, as determined by cell-based phosphorylation assays well known in the art, e.g., cell-based phosphorylation assays described herein.

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein inhibits at a 50% concentration (IC) of less than about 600pM, or less than about 500pM, or less than about 250pM ₅₀ ) Inhibition of KIT activity, such as ligand-induced tyrosine phosphorylation of KIT cytoplasmic domains, as determined by cell-based phosphorylation assays well known in the art, e.g., cell-based phosphorylation assays described herein. In a specific embodiment, the IC ₅₀ Less than about 550pM or 200pM. In particular embodiments, an IC ₅₀ Is in the range of about 50pM to about 225pM, or in the range of 100pM to about 600 pM. In particular embodiments, an IC ₅₀ In the range of about 50pM to about 550pM, or about 50pM to about 600pM, or about 150pM to about 550 pM.

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

In other embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein immunospecifically binds to a monomeric form of KIT (e.g., human KIT). In particular embodiments, an anti-KIT antibody provided herein, or an antigen-binding fragment thereof, specifically binds to a monomeric form of KIT (e.g., human KIT). In particular embodiments, an anti-KIT antibody provided herein, or an antigen-binding fragment thereof, specifically binds to a dimeric form of KIT (e.g., human KIT).

In particular embodiments, the anti-KIT antibodies or antigen-binding fragments thereof provided herein do not bind to monomeric forms of KIT and specifically bind to dimeric forms of KIT or multimeric forms of KIT. In certain embodiments, the antibody has a higher affinity for KIT monomers than for KIT dimers. In certain embodiments, the antibody has a higher affinity for KIT monomers than for KIT multimers.

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

In certain embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to human KIT or fragment thereof (e.g., the D4 region of human KIT) and specifically binds to canine (dog), feline (cat) and cynomolgus KIT, but does not specifically bind to murine or rat KIT or fragment thereof (e.g., the D4 region of murine KIT).

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to human KIT or fragment thereof (e.g., the D4 region of human KIT) and specifically to canine (dog), feline (cat) and cynomolgus KIT with higher affinity (e.g., at least 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold) than murine or rat KIT or fragment thereof (e.g., the D4 region of murine KIT).

In certain embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to an 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 repeated (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, and describes KIT mutations).

In certain embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein specifically binds to the extracellular domain of glycosylated human KIT. In certain embodiments, the antibodies or antigen binding fragments thereof described herein bind to 2 different glycosylated forms of the extracellular domain of human KIT. For example, 2 human KIT forms with different molecular weights have been observed by immunoblotting, indicating different glycosylation patterns.

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

In particular embodiments, an anti-KIT antibody or antigen-binding fragment thereof provided herein is a bivalent, monospecific antibody, wherein it has 2 antigen-binding regions (e.g., 2 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 VH and VL CDRs as shown in table 1. In a specific embodiment, the antigen binding region comprises a VH comprising the amino acid sequence of SEQ ID NO:8-12, comprising the amino acid sequence set forth in any one of SEQ ID NOs: 13-16. In certain aspects, the anti-KIT antibodies or antigen-binding fragments thereof provided herein are not dual-specific antibodies.

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

In particular embodiments provided herein, recombinant antibodies, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant, combinatorial antibody libraries, or antibodies prepared, expressed, produced, or isolated by any other means, including, for example, via synthesis, genetic engineering of DNA sequences encoding human immunoglobulin sequences, or splicing of human immunoglobulin gene sequences with other such sequences, can be isolated, produced, expressed, or produced by recombinant means. In certain embodiments, the amino acid sequences of these recombinant antibodies have been modified such that the amino acid sequences of these antibodies, e.g., VH and/or VL regions, are non-naturally occurring sequences within a germline repertoire of biological antibodies, e.g., within a murine or human germline repertoire. In particular embodiments, the recombinant antibody can be obtained by assembling some sequence fragments naturally occurring in an organism (e.g., primate, such as human) into a composite sequence of the recombinant antibody, wherein the composite sequence does not naturally occur within the organism (e.g., primate, such as human).

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 IgA 2), or subclass. In particular embodiments, the antibodies provided herein are IgG antibodies (e.g., human IgG antibodies) or classes (e.g., human IgG1 or IgG 4) or subclasses thereof. In another embodiment, the antibody described herein is an IgG (e.g., human IgG1 (isotype a, z or f)) or IgG4 antibody. In certain embodiments, the antibodies described herein are whole or whole antibodies, e.g., whole or whole humanized, human, or complex human antibodies.

In particular aspects, antibodies provided herein comprise antibody light and heavy chains, e.g., isolated light and heavy chains. In particular embodiments, the light chain of an antibody described herein is a kappa light chain relative to a light chain. In another embodiment, the light chain of an antibody described herein is a lambda light chain. In another embodiment, the light chain of an antibody described herein is a human kappa light chain or a human lambda light chain. In particular embodiments, the antibodies described herein comprise a human light chain constant region. Non-limiting examples of human light chain constant region sequences have been described in the art, see, for example, U.S. Pat. No.5,693,780 and Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5 th edition, U.S. Pat. No. of Health and Human Services, NIH Publication No.91-3242.

In particular embodiments, the heavy chain of an antibody described herein can be an alpha (α), delta (δ), epsilon (epsilon), gamma (gamma), or mu (mu) heavy chain, relative to the heavy chain. In another embodiment, the heavy chain of the antibody may comprise a human α (α), δ (δ), ε (ε), γ (γ), or μ (μ) heavy chain. In particular embodiments, antibodies described herein comprise a human heavy chain constant region (e.g., a human IgG constant region, e.g., a human IgG1, igG2, igG3, or IgG4 constant region). Non-limiting examples of human heavy chain constant region sequences have been described in the art, see, for example, U.S. Pat. No.5,693,780 and Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5 th edition, U.S. Pat. No. of Health and Human Services, NIH Publication No.91-3242. In particular embodiments, the antibodies described herein comprise a modified (e.g., mutated) human Fc region or domain (e.g., a modified (e.g., mutated) human IgG1 Fc region or domain, a modified (e.g., mutated) human IgG2 Fc region or domain, a modified (e.g., mutated) human IgG3 Fc region or domain, or a modified (e.g., mutated) human IgG4 Fc region or domain.

In certain embodiments, the anti-KIT antibodies described herein are human, composite human, or humanized monoclonal antibodies. In particular embodiments, the antibodies described herein are engineered antibodies, e.g., antibodies produced by recombinant methods. In particular embodiments, the antibodies described herein are humanized antibodies comprising one or more non-human (e.g., rodent or murine) CDRs and one or more human Framework Regions (FR), and optionally human heavy chain constant regions and/or light chain constant regions. In particular embodiments, the antibodies described herein comprise one or more primate (or non-human primate) framework regions. In particular embodiments, the antibodies described herein do not include a non-human primate framework region.

Antibodies provided herein can include antibodies comprising chemical modifications, e.g., antibodies that have been chemically modified, e.g., by covalently linking any type of molecule to the antibody. For example, but not by way of limitation, anti-KIT antibodies may be glycosylated, acetylated, pegylated, phosphorylated, or amidated, may be derivatized via a protecting/blocking group, or may further comprise a cellular ligand and/or other proteins or peptides (e.g., heterologous proteins or peptides), and the like. For example, the antibodies provided herein can be chemically modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, and the like. In addition, the anti-KIT antibodies described herein may contain one or more atypical amino acids.

In one embodiment, an anti-KIT antibody provided herein is a naked antibody that is not linked, fused or conjugated (e.g., artificially linked, fused or conjugated) to another molecule, peptide or polypeptide (e.g., a heterologous polypeptide). In particular embodiments, the anti-KIT antibodies provided herein are not antibody-drug conjugates. In particular embodiments, the anti-KIT antibodies provided herein are not fusion proteins. In particular embodiments, the anti-KIT antibodies described herein do not comprise any atypical amino acids.

5.1.1 antibody conjugates

In some embodiments, provided herein are antibodies (e.g., human or humanized antibodies) or antigen binding fragments thereof conjugated or recombinantly fused to a diagnostic, detectable, or therapeutic agent or any other molecule. Conjugated or recombinantly fused antibodies may be useful, for example, for monitoring or prognosticating the onset, progression, progress, and/or severity of a KIT-associated disorder or disease, e.g., as part of a clinical test procedure, such as determining the efficacy of a particular therapy. Conjugated or recombinantly fused antibodies may be useful, for example, for protecting, treating, or controlling KIT-related disorders, or for protecting, treating, or controlling the effects of KIT-related disorders. 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, e.g., stability (e.g., stability in serum), half-life, solubility, and antigenicity.

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

Such diagnosis and detection may be accomplished, for example, by coupling the antibody to a detectable molecule or substance, including but not limited to, a variety of enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-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 isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials such as (but not limited to) luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin or aequorin; radioactive materials, such as but not limited to iodine @, for example ¹³¹ I、 ¹²⁵ I、 ¹²³ I and ¹²¹ i) The carbon is ¹⁴ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹¹⁵ In、 ¹¹³ In、 ¹¹² In and ¹¹¹ in, technetium ] ⁹⁹ Tc), thallium ²⁰¹ Ti, ga ] ⁶⁸ Ga、 ⁶⁷ Ga and Pd% ¹⁰³ Pd and molybdenum% ⁹⁹ Mo and xenon ¹³³ Xe and F ¹⁸ F)、 ¹⁵³ Sm、 ¹⁷⁷ Lu、 ¹⁵⁹ Gd、 ¹⁴⁹ Pm、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁶⁶ Ho、 ⁹⁰ Y、 ⁴⁷ Sc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁴² Pr、 ¹⁰⁵ Rh、 ⁹⁷ Ru、 ⁶⁸ Ge、 ⁵⁷ Co、 ⁶⁵ Zn、 ⁸⁵ Sr、 ³² P、 ¹⁵³ Gd、 ¹⁶⁹ Yb、 ⁵¹ Cr、 ⁵⁴ Mn、 ⁷⁵ Se、 ¹¹³ Sn or ¹¹⁷ Sn; and positron-emitting metals using a variety of positron emission tomography, and nonradioactive paramagnetic metal ions.

Antibodies or antigen-binding fragments thereof conjugated or recombinantly fused to a therapeutic moiety (or one or more therapeutic moieties) as described herein are provided And uses of these antibodies. The antibodies can be conjugated or recombinantly fused to a therapeutic moiety, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent, or a radiometal ion, e.g., an alpha emitter. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Therapeutic moieties include, but are not limited to, auristatin or derivatives thereof, e.g., 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-disturbing agents, e.g., maytansine or derivatives thereof, e.g., maytansine DM1 (see, e.g., U.S. Pat. nos. 7,851,432, 7,575,748 and 5,416,064, each of which is incorporated herein by reference); prodrugs, for example, prodrugs of CC-1065 (rapamycin) analogs; antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil amamide); alkylating agents (e.g., nitrogen mustard, chlorambucil (thioepa chlorambucil), melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide (cyclophosphamide), busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP) and cisplatin); minor groove binding alkylating agent; anthracyclines (e.g., daunorubicin (previously known as daunorubicin) and doxorubicin); antibiotics (e.g., d-actinomycin (previously known as actinomycin), bleomycin, plicamycin, and Aflatoxin (AMC)); orientine molecules (e.g., orientine PHE, bryostatin 1, and sorastatin 10; see Woyke et al, antimicrob. Agents chemotherIt. 46:3802-8 (2002), woyke et al, antimicrob. Agents chemotherIt. 45:3580-4 (2001), mohammad et al, antimicer 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, progesterone, androgens and estrogens), DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarji) an et al, clin Cancer Res.8 (7): 2167-76 (2002)); cytotoxic agents (e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax-emitting diones, mitoxantrone, procamycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof and those disclosed in U.S. Pat. nos. 6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459, the disclosures of each of which are incorporated herein by reference); farnesyl transferase inhibitors (e.g., R115777, BMS-214662 and those disclosed by, for example, U.S. patent 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, 6,387,905, 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, 6,211,193, 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, the disclosures of which are each incorporated herein by reference); topoisomerase inhibitors (e.g., camptothecin, irinotecan, SN-38, topotecan, 9-aminocamptothecin, GG-211 (GI 147211), DX-8951f, IST-622, lubitecan, pyrazoline acridine, XR-5000, sainitopin, UCE6, UCE1022, TAN-1518A;TAN 1518B;KT6006;KT6528;ED-110, NB-506, ED-110, NB-506, and butterfly mycin); bragg factor (bulgarein); DNA minor groove binders such as Hoescht dye 33342 and Hoechst dye 33258; two sides Needle alkali; faguronin; epiberberine; ke Nan factor; beta-lapachone; BC-4-1; bisphosphonates (e.g., amitraz phosphate, cimadronate, clodronate, tiludronate, oxyethyldiphosphate, ibandronate, neridronate, olpandronate, risedronate, piridronate, pamidronate, zolbehenate), HMG-CoA reductase inhibitors (e.g., lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, inhibin, cerivastatin, lyxol), lipstatin (lupipe), 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); itemozolomide tetan (tiuxetan)Toximomab->) And pharmaceutically acceptable salts, solvates, clathrates and prodrugs thereof.

In a specific embodiment, the therapeutic moiety or drug moiety is an anti-tubulin drug, such as auristatin or a derivative thereof. Non-limiting examples of auristatins 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, e.g., 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, for example, a prodrug of a CC-1065 (rachelmycin) analog (see, e.g., U.S. patent application publication No.2008/0279868 and PCT international patent application publications nos. WO 2009/017394, WO 2010/062171, and WO 2007/089149), each of which are incorporated herein by reference.

In particular embodiments, the antibody and therapeutic/pharmaceutical agent are conjugated through one or more linkers. In another embodiment, the antibody and therapeutic/pharmaceutical agent are conjugated directly.

In particular embodiments, non-limiting examples of therapeutic or pharmaceutical moieties for conjugation to antibodies described herein include calicheamicin (e.g., LL-E33288 complex, e.g., γ -calicheamicin (calicheamicin), see, e.g., U.S. patent No.4,970,198) and derivatives thereof (e.g., γ -calicheamicin (calicheamicin) hydrazide derivatives), ozamicin (ozigamicins), a carcinotoxin and derivatives thereof (e.g., CC-1065 (NSC 298223) or achiral analogs of carcinotoxin (e.g., AS-1-145 or stanamycin)), 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 2006/031, WO 089809, WO 2005/0807 and WO 2005/9808 are incorporated herein by reference in their entirety.

Non-limiting examples of calicheamicins (calicheamicins) suitable for conjugation to antibodies described herein are disclosed, for example, in U.S. patent No.4,671,958;5,053,394;5,037,651;5,079,233; and 5,108,912; and PCT international patent application publications nos. WO 2011/021146, WO 2008/150261, WO 2006/031653, WO 2005/089809, WO 2005/089807 and WO 2005/089808; for the disclosure of calicheamicin, each of the above patents is incorporated herein by reference. In particular embodiments, these compounds may contain methyl trisulfide that reacts with the appropriate thiol to form disulfide, and at the same time introduce functional groups, such as hydrazides or other functional groups that may be used to conjugate calicheamicin (calicheamicin) to antibodies described herein. In certain embodiments, improved antibody/drug conjugates can be obtained by adding a dimethyl substituent to stabilize disulfide bonds present in calicheamicin (calicheamicin) conjugates. In a specific embodiment, the calicheamicin derivative is N-acetyl gamma calicheamicin dimethyl hydrazide or NAc-gamma DMH (CL-184,538), which is one of the derivatives optimized for conjugation. Disulfide analogs of calicheamicin that can be conjugated to antibodies described herein are described, for example, in U.S. Pat. nos. 5,606,040 and 5,770,710, each of which is incorporated herein by reference for the disclosure of such compounds. In certain embodiments, a moiety (e.g., calicheamicin or a derivative thereof) is conjugated to an antibody through a linker. In particular embodiments, a moiety (e.g., calicheamicin or a derivative thereof) is hydrolyzed from the antibody-drug conjugate at the linker. In one embodiment, the hydrolysis moiety (e.g., calicheamicin or a derivative thereof) from the antibody conjugate at the junction is at a temperature of 20 to 50 ℃, preferably 37 ℃, between about pH 3.0 and pH 4.0 for 1-24 hours.

In particular embodiments, non-limiting examples of therapeutic or pharmaceutical moieties for conjugation to antibodies described herein include Pyrrolobenzodiazepine (PBD) and derivatives thereof, e.g., PBD dimers (e.g., SJG-136 or SG 2000), C2-unsaturated PBD dimers, pyrrolobenzodiazepine dimers with C2 aryl substitutions (e.g., SG 2285), PBD dimer prodrugs activated by hydrolysis (e.g., SG 2285) and polypyrrole-PBDs (e.g., SG 2274) (see, e.g., PCT international patent application publication nos. WO 2000/0126507, WO 2007/039752, WO 2005/110423, WO 2005/085251 and WO 2005/040170, the disclosure of which is incorporated herein by reference, and U.S. patent No.7,612,062).

In addition, the antibodies described herein can be conjugated to a therapeutic moiety, such as a radioactive metal ion, such as an alpha emitter, such as ²¹³ Bi, or for the incorporation of radioactive metal ions, including but not limited to ¹³¹ In、 ¹³¹ LU、 ¹³¹ Y、 ¹³¹ Ho、 ¹³¹ Sm is conjugated to a macrocyclic chelator of polypeptides. In certain embodiments, the macrocyclic chelating agent is 1,4,7, 10-tetraazateHeterocyclododecane-N, N' -tetraacetic acid (DOTA), which may be linked to an antibody through a linker molecule. These linker molecules are generally known in the art and are described in Denardo et al, 1998,Clin Cancer Res.4 (10): 2483-90; peterson et al 1999, bioconjug. Chem.10 (4): 553-7; and Zimmerman et al 1999, nucl. Med. Biol.26 (8): 943-50, each of which is incorporated by reference in its entirety.

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

In certain embodiments, the moiety is conjugated to the antibody through one or more linkers. In a specific embodiment, a moiety is hydrolyzed from the antibody-drug conjugate at the linker. In one embodiment, the moiety is hydrolyzed from the antibody conjugate at the linker for about 1-24 hours at a temperature of about 20 to 50 ℃, preferably 37 ℃, between about pH 3.0 and pH 4.0. In particular embodiments, the linker is stable in the blood stream, but once inside the target cell, the conjugate moiety is released. In certain embodiments, moieties are conjugated to antibodies described herein via one or more triazole-containing linkers (see, e.g., international patent application publication No. wo 2007/018431, incorporated herein by reference). Non-limiting examples of linkers and spacers for incorporating 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.

Furthermore, the antibodies described herein may be fused to a marker sequence, such as a peptide, to aid in purification. In a preferred embodiment, the marker amino acid sequence is a hexahistidine peptide, such as a tag provided in a pQE vector (QIAGEN, inc.), and the like, many of which are commercially available. As described in Gentz et al, 1989,Proc.Natl.Acad.Sci.USA 86:821-824, for example, hexahistidine provides for convenient purification of fusion proteins. Other peptide tags used for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin protein (Wilson et al, 1984, cell 37:767) and "FLAG" tags.

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 (Main plaited), pages 243-56 (Alan R.Lists, inc. 1985); hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2 nd edition), robinson et al (Main plaited), pages 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 (Main plaited), pages 475-506 (1985); "Analysis, results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, baldwin et al (Main plaited), pages 303-16 (Academic Press 1985), thorpe et al, 1982, immunol. Rev.62:119-58; U.S. Pat. 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 patent publications WO 91/06570, WO 96/04388, WO96/22024, 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, the entire contents of which are incorporated herein by reference.

The antibodies described herein may also be attached to a solid support, which is particularly useful for immunoassay or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride, or polypropylene.

In a certain aspect, an antibody or antigen binding fragment thereof described herein is an extracellular drug conjugate (ECD) comprising an antibody optionally linked to a drug by a linker (see, e.g., PCT international patent application publication No. wo 2011/031870). The drug may act extracellular and thus does not require internalization of the conjugate. After the ECD binds to the target cell, the drug signals the cell.

In one embodiment, the linker of the ECD is a non-cleavable linker. Examples of non-cleavable linkers include linkers that contain polyethylene glycol chains or polyethylene chains that are insensitive to acids or bases (e.g., hydrazone-containing linkers), linkers that are insensitive to reducing or oxidizing agents (e.g., those that contain disulfide bonds), and linkers that are insensitive to enzymes that may be present in the cell or circulatory system. Specific examples of non-cleavable linkers include SMCC linkers (U.S. patent application 20090202536). For illustrative purposes, examples of cleavable linkers include linkers containing unhindered glutathione-sensitive disulfides, esters, peptide sequences sensitive to peptidases such as cathepsins or plasmins, pH-sensitive hydrazones (see Bioconjugate chem.,2010,21 (1), pages 5-13), and unhindered disulfide linkers SPP (U.S. patent application 20090202536).

In certain aspects, the ECD comprises a drug or agent that is a cardiac glycoside, e.g., a eleutherobin or a sugar-enhanced eleutherobin. In one embodiment, the agent consists of a sugar-free cardiac glycoside. In various embodiments, the cardiac glycoside is a compound identified in PCT patent publication No. wo 2010/017480 (PCT/US 2009/053159).

5.2 Polynucleotide

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., light chain variable regions and/or heavy chain variable regions) described herein that immunospecifically bind to a KIT antigen. Also provided herein are polynucleotides encoding KIT antigens for use in the production of anti-KIT antibodies described herein.

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 that are present in the natural source of the nucleic acid molecule (e.g., in a human). In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or may be substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes preparation of polynucleotides or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (specifically less than about 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and/or other chemicals. In particular embodiments, nucleic acid molecules encoding antibodies described herein are isolated or purified.

In a particular aspect, provided herein are polynucleotides or combinations of polynucleotides comprising nucleotide sequences encoding the antibodies or antigen-binding fragments thereof described herein or VH and VL of the antibodies or antigen-binding fragments thereof. In particular embodiments, provided herein are nucleic acid sequences comprising a VH encoding an antibody described herein, or an antigen-binding fragment thereof. In particular embodiments, provided herein are nucleic acid sequences comprising a VL encoding an antibody described herein, or an antigen binding fragment thereof. In particular embodiments, provided herein are polynucleotides comprising a first nucleotide sequence encoding a VH of an antibody described herein, or an antigen-binding fragment thereof, and a second nucleotide sequence encoding a VL of an antibody described herein. In particular embodiments, provided herein are combinations of two polynucleotides, wherein a first polynucleotide of the combination comprises a first nucleotide sequence encoding a VH or antigen-binding fragment thereof of an antibody described herein, and a second polynucleotide of the combination comprises a second nucleotide sequence encoding a VL or antigen-binding fragment thereof of the antibody. In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding the heavy chain of an antibody described herein. In particular embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain of an antibody described herein. In particular embodiments, provided herein are polynucleotides 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 particular embodiments, provided herein are combinations of two polynucleotides, wherein a first polynucleotide of the combination comprises a first nucleotide sequence encoding a heavy chain of an antibody described herein, and a second polynucleotide of the combination comprises a second nucleotide sequence encoding a light chain of the antibody.

In particular embodiments, the polynucleotides provided herein comprise SEQ ID NOs: 23. In particular embodiments, the polynucleotides provided herein comprise SEQ ID NOs: 24, and a nucleotide sequence shown in seq id no. In particular embodiments, the polynucleotides provided herein comprise SEQ ID NOs: 23 and SEQ ID NO:24, and a nucleotide sequence shown in seq id no. In particular embodiments, a combination of polynucleotides provided herein comprises: comprising SEQ ID NO:23 and a first polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:24, and a second polynucleotide of the nucleotide sequence shown in seq id no.

Also provided herein are polynucleotides encoding optimized anti-KIT antibodies or fragments thereof, e.g., by codon/RNA optimization, heterologous signal sequence substitution, and removal of mRNA instability elements. Thus, it is possible to use, for example, U.S. Pat. No.5,965,726;6,174,666;6,291,664;6,414,132; and 6,794,498 by introducing codon changes and/or eliminating inhibitory regions in the mRNA, thereby producing an optimized nucleic acid encoding an anti-KIT antibody or fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression. For example, potential splice sites and labile elements (e.g., A/T or A/U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. The changes use degeneracy of the genetic code, e.g., substitution codons using the same amino acids. In some embodiments, it may be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid having a similar chemical structure and properties and/or function as the original amino acid. These methods can increase the expression of an anti-KIT antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to the expression of an anti-KIT antibody encoded by an unoptimized polynucleotide.

In certain embodiments, an optimized polynucleotide sequence encoding an anti-KIT antibody or fragment thereof (e.g., VL domain and/or VH domain) described herein may hybridize to an antisense (e.g., complement) polynucleotide encoding an unoptimized polynucleotide sequence of an anti-KIT antibody or fragment thereof (e.g., VL domain and/or VH domain) described herein. In particular embodiments, an optimized nucleotide sequence encoding an anti-KIT antibody or fragment described herein hybridizes under high stringency conditions to an antisense polynucleotide encoding an unoptimized polynucleotide sequence of an anti-KIT antibody or fragment described herein. In particular embodiments, an optimized nucleotide sequence encoding an anti-KIT antibody or fragment thereof described herein hybridizes under high, medium, or low stringency hybridization conditions to an antisense polynucleotide encoding an non-optimized nucleotide sequence of an anti-KIT antibody or fragment thereof described herein. Information about hybridization conditions has been described, see, for example, U.S. patent application publication No. US2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.

In certain embodiments, the optimized polynucleotide sequence encoding the VL region of an antibody described herein hybridizes to SEQ ID NO:23, has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity. In certain embodiments, the optimized polynucleotide sequence encoding the VH region of the antibodies described herein hybridizes to SEQ ID NO:24, has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity.

Polynucleotides may be obtained and the nucleotide sequence of the polynucleotides determined by any method known in the art. The nucleotide sequences encoding the antibodies described herein, as well as modified versions of these antibodies, can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way as to produce nucleic acids encoding the antibodies. Such polynucleotides encoding the antibodies may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, 1994,BioTechniques 17:242), which briefly include synthesis of overlapping oligonucleotides comprising portions of the sequences encoding the antibodies, annealing of those oligonucleotides, and ligation, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, polynucleotides encoding antibodies described herein can be produced from nucleic acids from suitable sources (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3 'and 5' ends of known sequences can be performed using genomic DNA from hybridoma cells producing the antibody of interest. These PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding the light and/or heavy chains of an antibody. These PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding the light chain variable region and/or the heavy chain variable region of an antibody. The amplified nucleic acid may be cloned into a vector for expression in a host cell and for further cloning, e.g., to produce chimeric and humanized antibodies.

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

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 specifically binding 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 may be placed in an expression vector and then transfected into a host cell, such as an E.coli (E.coli) cell, simian COS cell, chinese Hamster Ovary (CHO) cell (e.g., from the CHO GS System) ^TM (Lonza) CHO cells) or myeloma cells that do not otherwise produce immunoglobulins, to obtain synthesis of anti-KIT antibodies in recombinant host cells.

To generate complete antibodies, PCR primers can be used to amplify VH or VL sequences in scFv clones, including VH or VL nucleotide sequences, restriction sites, and flanking sequences that protect the restriction sites. The PCR amplified VH domain may be cloned into a vector expressing a heavy chain constant region, e.g., a human γ1 or γ4 constant region, and the PCR amplified VL domain may be cloned into a vector expressing a light chain constant region, e.g., a human kappa or lambda constant region, using cloning techniques known to those skilled in the art. In certain embodiments, a vector for expressing a VH or VL domain comprises an EF-1 a promoter, a secretion signal, a cloning site for a variable domain, a constant domain, and a selectable marker, such as neomycin. The VH and VL domains may also be cloned into a vector expressing the necessary constant regions. The heavy chain and light chain transfer vectors are then co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing full length antibodies, e.g., igG.

The DNA may also be modified, for example, by: the human heavy and light chain constant domains are replaced with coding sequences to replace murine sequences, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

5.3 recombinant expression of host cells and antibodies

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

Recombinant expression of an antibody (e.g., a full length antibody, heavy and/or light chain of an antibody, or a single chain antibody as described herein) that immunospecifically binds to a KIT antigen as described herein includes construction of an expression vector comprising a polynucleotide encoding the antibody. Once the polynucleotides encoding antibody molecules, heavy and/or light chains of antibodies, or fragments thereof (preferably, but not necessarily, containing heavy and/or light chain variable domains) described herein have been obtained, vectors for antibody molecule production can be produced by DNA recombination techniques using techniques well known in the art. Thus, described herein are methods for producing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies (or VH/VL or heavy/light chains). Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, recombinant DNA techniques in vitro, synthetic techniques, and genetic recombination in vivo. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a fragment thereof, or a heavy or light chain CDR, as described herein, operably linked to a promoter. These vectors may, for example, comprise nucleotide sequences encoding the constant regions of an antibody molecule (see, e.g., international patent publication Nos. WO 86/05807 and WO 89/01036; and U.S. Pat. No.5,122,464), and the variable domains of the antibodies may be cloned into the vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

In particular embodiments, provided herein are vectors comprising a polynucleotide encoding a VH of an antibody described herein, or an antigen-binding fragment thereof. In particular embodiments, provided herein are vectors comprising polynucleotides encoding the VL of the antibodies described herein or antigen binding fragments thereof. In particular embodiments, provided herein are vectors comprising polynucleotides encoding VH and VL of the antibodies described herein, or antigen-binding fragments thereof. In particular embodiments, provided herein are vectors comprising a first polynucleotide encoding a VH of an antibody or antigen-binding fragment thereof described herein, and a second polynucleotide encoding a VL of the antibody or antigen-binding fragment. In particular embodiments, provided herein are combinations of two vectors, wherein a first vector of the combination comprises a first polynucleotide encoding a VH of an antibody or antigen-binding fragment thereof described herein, and a second vector of the combination comprises a second polynucleotide encoding a VL of the antibody or antigen-binding fragment. In particular embodiments, provided herein are vectors comprising polynucleotides encoding the heavy chains of antibodies described herein. In particular embodiments, provided herein are vectors comprising polynucleotides encoding the light chains of antibodies described herein. In particular embodiments, provided herein are vectors comprising polynucleotides encoding the heavy and light chains of the antibodies described herein. In particular embodiments, provided herein are vectors comprising a first polynucleotide encoding a VH of an antibody described herein and a second polynucleotide encoding a VL of the antibody. In particular embodiments, provided herein are combinations of two vectors, wherein a first vector of the combination comprises a first polynucleotide encoding a VH of an antibody described herein, and a second vector of the combination comprises a second polynucleotide encoding a VL of the antibody.

The expression vector or combination of expression vectors can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce the antibodies or fragments thereof described herein. Thus, provided herein are host cells containing a polynucleotide or combination of polynucleotides encoding an antibody or fragment thereof, or heavy or light chain or fragment thereof, as described herein, or a single chain antibody or polynucleotide as described herein, operably linked to a promoter for expression of these sequences in the host cells. In certain embodiments, for expression of diabodies, vectors encoding both heavy and light chains may be co-expressed alone in a host cell to express the entire immunoglobulin molecule, as described in detail below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding both the heavy and light chains (or both VH and VL) or fragments thereof of an antibody described herein. In particular embodiments, the host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain (or VH) or fragment thereof of an antibody described herein, and a second vector comprising a polynucleotide encoding a light chain (or VL) or fragment thereof of an antibody described herein. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain (or VH) or fragment thereof of an antibody described herein, and the second host cell comprises a second vector comprising a polynucleotide encoding a light chain (or VL) or fragment thereof of an antibody described herein.

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 by which the coding sequences of interest can be produced and subsequently purified, and also represent cells that can express the antibody molecules described herein in situ when transformed or transfected with suitable nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences (e.g., e.coli (e. Coli) and bacillus subtilis (b); yeast transformed with recombinant yeast expression vectors containing antibody coding sequences (e.g., pichia pastoris (Saccharomyces pichia)); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; recombinant virus expression vector containing antibody coding sequenceFor example, cauliflower mosaic virus, caMV; tobacco mosaic virus, TMV) or plant cell systems (e.g., green algae, such as chlamydomonas reinhardtii (Chlamydomonas reinhardtii)) infected with recombinant plasmid expression vectors (e.g., ti plasmids) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, MDCK, HEK 293, NS0, per.c6, VERO, CRL7O3O, hsS Bst, heLa and NIH 3T3 cells) having recombinant expression constructs comprising a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; vaccinia virus 7.5K promoter). In particular embodiments, the cells used to express the antibodies or antigen-binding fragments thereof described herein are CHO cells, e.g., from CHO GS System ^TM (Lonza) CHO cells. In a specific embodiment, the mammalian expression vector is pOptiVEC ^TM Or pcDNA3.3. Preferably, bacterial cells, such as e.coli (Escherichia coli), and more preferably eukaryotic cells, in particular eukaryotic cells for expression of the whole recombinant antibody molecule, are used for expression of the recombinant antibody molecule. For example, mammalian cells, such as Chinese Hamster Ovary (CHO) cells, are effective antibody expression systems in combination with vectors derived from human cytomegalovirus, such as the major intermediate early gene promoter element (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 particular embodiments, expression of a nucleotide sequence encoding an antibody described herein that immunospecifically binds to a KIT antigen is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, some expression vectors may be advantageously selected based on the use of antibody molecules intended for expression. For example, when large amounts of such antibodies are produced, vectors directing high-level expression of fusion protein products that are readily purified may be desirable for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, the E.coli (E.coli) expression vector pUR278 (Ruther et al, 1983,EMBO 12:1791), to which antibody coding sequences can be separately ligated in frame with the lac Z coding region, thereby producing fusion proteins; pIN vector (Inouye & Inouye,1985,Nucleic Acids Res.13:3101-3109;Van Heeke&Schuster,1989,J.Biol.Chem.24:5503-5509); etc. pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, these fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione sepharose beads, followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In insect systems, the alfalfa silver vein moth (Autographa californica) nuclear polyhedrosis virus (AcNPV) was used as a vector to express exogenous genes. The virus grows in spodoptera frugiperda (Spodoptera frugiperda) cells. Antibody coding sequences can be cloned separately into an unnecessary region of the virus (e.g., the polyhedrin gene) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, some viral-based expression systems may be used. If an adenovirus is used as an expression vector, the antibody coding sequence of interest may be linked to an adenovirus transcription/translation control complex, e.g., a late promoter and a tripartite leader sequence. Such chimeric genes can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in an unnecessary region of the viral genome (e.g., the El or E3 region) will result in the production of a recombinant virus that survives the infected host and is capable of expressing the antibody molecule (see, e.g., logan & Shenk,1984,Proc.Natl.Acad.Sci.USA 8 1:355-359). For efficient translation of inserted antibody coding sequences, a specific initiation signal may also be required. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be synchronized with the reading frame of the desired coding sequence (in phase with) to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. Expression efficiency may be increased by the inclusion of suitable transcription enhancer elements, transcription terminators, and the like (see, e.g., bittner et al, 1987,Methods in Enzymol.153:51-544).

Alternatively, host cell lines may be selected that regulate expression of the inserted sequences, or alter and process the gene product in a specific manner as desired. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems may be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with the cellular mechanisms for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. These mammalian host cells include, but are not limited to, CHO, VERO, BHK, hela, COS, MDCK, HEK 293, NIH 3T3, W138, BT483, hs578T, HTB, BT2O and T47D, NS0 (murine myeloma cell lines that do not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells. In certain embodiments, the humanized monoclonal anti-KIT antibodies described herein are produced in mammalian cells, such as CHO cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing antibody molecules may be engineered. Instead of using an expression vector containing a viral origin of replication, the host cell may be transformed with DNA controlled by suitable expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. Following the introduction of the exogenous DNA, the engineered cells may be allowed to grow in the enrichment medium for 1-2 days and then transferred to the selection medium. Selectable markers in recombinant plasmids confer selection tolerance and allow cells to stably integrate the plasmids into their chromosomes and grow to form points (foci) which in turn can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the antibody molecules. These engineered cell lines may be particularly useful in the screening and evaluation of compositions that interact directly or indirectly with antibody molecules.

Some selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al, 1977, cell 11:223), xanthine guanine phosphoribosyl transferase (Szybalska)&Szybalski,1992,Proc.Natl.Acad.Sci.USA 48:202) and adenine phosphoribosyl transferase (Lowy et al, 1980, cell 22:8-17) genes, which can be used in tk-, hgprt-or aprt-cells, respectively. In addition, antimetabolite tolerance can be used as a basis for selection of the following genes: dhfr, which confers tolerance 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 tolerance to mycophenolic acid (Mulligan&Berg,1981,Proc.Natl.Acad.Sci.USA 78:2072); neo, which confers tolerance to the aminoglycoside class G-418 (Wu and Wu,1991,Biotherapy 3:87-95; tolstoshov, 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): l55-2 15); and hygro, which confers tolerance to hygromycin (Santerre et al, 1984, gene 30:147). The desired recombinant clone can be selected conventionally using methods generally known in the art of DNA recombination, and these methods are described, for example, in Ausubel et al (major code), CurrentProtocolsin MolecularBiology,John Wiley&Sons,NY(1993)；Kriegler,Gene TransferandExpressionA Laboratory Manual, stockton Press, NY (1990); and in chapters 12 and 13, dragopoli et al (main edition),Current Protocols in Human Genetics,John Wiley&sons, NY (1994); colberre-Garapin et al, 1981, J.mol. Biol.150:1, the entire contents of which are incorporated herein by reference.

Expression levels of antibody molecules can be increased by vector amplification (for reviews, see Bebbington and Hentschel, the use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, volume 3 (Academic Press, new York, 1987)). When the marker in the antibody expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with an antibody gene, antibody production will also increase (Crouse et al, 1983, mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two or more expression vectors described herein, wherein the first vector encodes a heavy chain-derived polypeptide and the second vector encodes a light chain-derived polypeptide. The two vectors may contain the same selectable marker that allows for equal expression of the heavy and light chain polypeptides. The host cell may be co-transfected with different amounts of two or more expression vectors. For example, the host cell may be transfected with any of the following ratios of first expression vector to second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

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

Once the antibody molecule described herein is produced by recombinant expression, it may be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, specifically by affinity and size exclusion column chromatography for specific antigens after protein a), centrifugation, differential solubility, or by any other standard technique for protein purification. Furthermore, antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to aid in purification.

In specific embodiments, the antibodies described herein are isolated or purified. Typically, an isolated antibody is an antibody that is substantially free of other antibodies having antigen specificity different from the isolated antibody. For example, in particular embodiments, the antibodies described herein are prepared substantially free of cellular material and/or chemical precursors. The language "substantially free of cellular material" includes the preparation of antibodies, wherein the antibodies are separated from cellular components of the cells from which the antibodies were isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparation of antibodies having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and/or variants of the antibodies, e.g., post-translational modified forms of the antibodies or other different forms of the antibodies (e.g., antibody fragments). When the antibody is recombinantly produced, it is also typically substantially free of culture medium, i.e., culture medium represents 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 typically substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that are involved in protein synthesis. Thus, these antibodies produce chemical precursors or compounds having less than about 30%, 20%, 10% or 5% (by dry weight) in addition to the antibody of interest. In particular embodiments, the antibodies described herein are isolated or purified.

5.4 antibody production

Antibodies (e.g., human or humanized antibodies) (or antigen-binding fragments thereof) that immunospecifically bind to a KIT antigen described herein can be produced by any method known in the art for antibody synthesis, e.g., by chemical synthesis or by recombinant expression techniques. In a particular aspect, provided herein are methods for producing an antibody described herein, comprising culturing a host cell described herein and/or expressing the antibody using a host cell described herein, optionally further comprising purifying the antibody obtained from the host cell. Unless otherwise indicated, the methods described herein employ techniques conventional in the relevant arts of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and 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, 2 nd edition, cold Spring Harbor Laboratory Press; sambrook et al (2001) Molecular Cloning: ALaboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY; ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons (1987 and yearly updates); current Protocols in Immunology, john Wiley & Sons (1987 and yearly updates); gait (Main plaited) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; eckstein (Main plaited) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; birren et al (Main plaited) (1999) Genome Analysis: A Laboratory Manual, cold Spring Harbor Laboratory Press.

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 patent publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), veneering (veneering) or surface remodeling (resurfacing) (European patent Nos. EP 592,106 and EP 519,596;Padlan,1991,Molecular Immunology28 (4/5): 489-498; studnica et al, 1994,Protein Engineering 7 (6): 805-814); and Roguska et al, 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No.5,565,332) and, for example, U.S. Pat. No.6,407,213, U.S. Pat. No.5,766,886, WO 9317105, tan et al, J.Immunol.169:1119 25 (2002), caldas et al, protein Eng.13 (5): 353-60 (2000), morea et al, methods 20 (3): 267 79 (2000), baca et al, J.biol. Chem.272 (16): 10678-84 (1997), roguska et al, protein Eng.9 (10): 895 904 (1996), couto et al, cancer Res.55 (23 support): 5973s-5977s (1995), couto et al, cancer Res.55 (8): 1717-22 (1995), pedfu JS 150 (409-10): 35 (1994), and Biol et al, 1993). See also U.S. patent publication No. US 2005/0042664Al (Feb.24, 2005), incorporated herein by reference.

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, hybridoma techniques may be used, including those known and taught in the art, e.g., harlow et al Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988); monoclonal antibodies are produced by those known and taught in Hammerling et al, in Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced by recombinant techniques, e.g., by expression of the recombinant monoclonal antibodies by a host cell, such as a mammalian host cell.

Methods for generating and screening specific antibodies using hybridoma technology are conventional and well known in the art. For example, in a hybridoma method, a mouse or other suitable host animal, such as sheep, goat, rabbit, rat, hamster, or cynomolgus monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization (e.g., the extracellular domain of human KIT). Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal Antibodies: principles and Practice, pages 59-103 (Academic Press, 1986)). In addition, RIMMS (multiple site repeat immunization) techniques may be used to immunize animals (Kilptrack et al, 1997Hybridoma 16:381-9, incorporated herein by reference).

Non-limiting examples of myeloma cell lines include murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors from Salk Institute Cell Distribution Center, san Diego, calif., USA, and SP-2 or X63-Ag8.653 cells from American type culture Collection (American Type Culture Collection), rockville, MD, USA. Human myeloma and mouse-human hybrid myeloma 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, pages 51-63 (Marcel Dekker, inc., new York, 1987)). [00157]Antibodies described herein include antibody fragments that recognize a specific KIT antigen, and the antibodies described herein can be produced by any technique known to those of skill in the art. For example, it is possible to use enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') ₂ Fragments) of immunoglobulin molecules to produce Fab and F (ab') ₂ Fragments. The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CHI domains of the heavy chain. F (ab') ₂ The fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds in the hinge region.

In one aspect, to generate an intact antibody, PCR primers comprising VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to clonally amplify VH or VL sequences from a template, e.g., scFv. The PCR amplified VH domain may be cloned into a vector expressing a VH constant region, and the PCR amplified VL domain may be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region, using cloning techniques known to those skilled in the art. The VH and VL domains may also be cloned into a vector expressing the necessary constant regions. The heavy chain and light chain transfer vectors are then co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing full length antibodies, e.g., igG.

Single domain antibodies, e.g., antibodies lacking a light chain, may 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.l (3): 253-263; muylederman, 2001, J. Iotechnol.74 (4): 277302; U.S. Pat. No.6,005,079; and International patent publication Nos. WO 94/04678, WO 94/25591 and WO 01/44301.

5.5 methods of treatment and medical uses

Provided herein are methods for impeding, preventing, protecting, treating, and/or controlling a KIT-related disorder or disease. These 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 also methods for preventing, impeding, protecting, treating, or controlling one or more symptoms of a KIT-related disorder or disease.

In particular embodiments, the methods described herein for treating a KIT-associated disorder or disease provide for a reduction or improvement in the development, severity, and/or duration of a KIT-associated disorder or disease due to administration of one or more therapies, including (but not limited to) administration of one or more prophylactic or therapeutic agents, such as anti-KIT antibodies described herein. In other embodiments, the methods described herein for treating a KIT-related disorder or disease involve a reduction in one or more symptoms of the KIT-related disorder or disease. In particular embodiments, the antibodies or antigen-binding fragments thereof or conjugates thereof described herein or the pharmaceutical compositions described herein are used to protect, treat, or control KIT-related disorders. In particular embodiments, a KIT-associated disease or disorder to be treated or controlled or protected using an anti-KIT antibody or antigen-binding fragment thereof or conjugate thereof described herein or a pharmaceutical composition described herein is associated with expression and/or activity of KIT, e.g., involves cells expressing KIT and/or exhibiting KIT activity, but is not caused or caused by expression or activity of KIT.

In particular embodiments, the antibody used in the methods described herein is internalized by the cell to which it binds. In particular embodiments, conjugates are used in the methods described herein, wherein the conjugates comprise an antibody described herein (e.g., a humanized anti-KIT antibody) or KIT-binding fragment thereof. In particular embodiments, the conjugate comprises an antibody described herein (e.g., a humanized anti-KIT antibody) or KIT-binding fragment thereof, covalently or non-covalently linked to a therapeutic agent, such as a toxin. In certain embodiments, the conjugate used in the methods described herein internalizes into the cell to which it binds.

In certain embodiments, the cell expresses KIT abnormally (e.g., highly), e.g., KIT is overexpressed. In particular embodiments, KIT expression (e.g., expression on the cell surface) is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than KIT expression on the surface of a control cell (e.g., a cell that expresses a normal level of KIT, e.g., a normal, e.g., human mast cell, stem cell, brain cell, melanocyte, or ovarian cell). In particular embodiments, KIT expression results in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher cell surface KIT expression than the average KIT expression on the surface of a control cell population (e.g., a cell population that expresses normal levels of KIT, e.g., a normal, e.g., human mast cell population, stem cell population, brain cell population, melanocyte population, or ovarian cell population). In particular embodiments, these control cells can be obtained or derived from a healthy individual (e.g., a healthy human). In some embodiments, KIT may be abnormally up-regulated in a particular cell type, whether or not KIT is abnormally expressed on the cell surface. In particular embodiments, KIT signaling or activity may be abnormally up-regulated in a particular cell type, whether or not KIT is abnormally expressed on the cell surface. In particular embodiments, KIT signal transduction is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than KIT signal transduction of a control cell (e.g., a cell containing normal KIT signal transduction, e.g., a mast cell, stem cell, brain cell, melanocyte, or ovarian cell). In particular embodiments, KIT signaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the average KIT signaling of a control cell population (e.g., a cell population exhibiting normal KIT signaling, e.g., a normal, e.g., human mast cell population, stem cell population, brain cell population, melanocyte population, or ovarian cell population). In certain embodiments, normal, abnormal or excessive cell signaling is caused by the binding of KIT to KIT ligand. In other embodiments, aberrant or excessive cell signaling occurs independent of KIT binding to KIT ligand.

In certain aspects, KIT-related disorders or diseases may be characterized by functionally acquired KIT activity, elevated KIT activity, or overexpression of KIT. In one embodiment, the KIT-associated disorder or disease is caused or caused, in whole or in part, by a functionally acquired KIT activity or expression of KIT, e.g., over-expression. In certain embodiments, the functionally acquired KIT activity may occur independently of KIT ligand (e.g., SCF) that binds to a KIT receptor. In particular aspects, high expression or overexpression of KIT in a cell refers to an expression level that is greater than the expression level of a reference cell known to have normal KIT expression or KIT activity or at least about 35%, 45%, 55%, or 65% greater than the average expression level in a population or sample of cells known to have normal KIT expression or KIT activity. In particular aspects, high expression or overexpression of KIT in a cell refers to an expression level that is greater than the expression level of a reference cell known to have normal KIT expression or KIT activity or at least about 35%, 45%, 55%, or 65% greater than the average expression level in a population or sample of cells known to have normal KIT expression or KIT activity. Expression levels of KIT may be assessed by methods described herein or known to those of skill in the art (e.g., western blot or immunohistochemistry). In particular embodiments, KIT-related disorders or diseases are characterized by KIT activity that is higher than normal KIT activity and that contributes to cell transformation, neoplasia and tumorigenesis. In particular aspects, high KIT activity or an increase in KIT activity in a cell refers to a level of KIT activity that is greater than the expression level of a reference cell known to have normal KIT activity or at least about 35%, 45%, 55% or 65% greater than the average level of KIT activity in a population or sample of cells known to have normal KIT activity. Non-limiting examples of KIT activity include tyrosine phosphorylation of cytoplasmic domains of KIT and signaling downstream of KIT, such as Stat or Akt signaling.

In certain embodiments, the KIT-associated disorder is a mast cell-associated disorder, eosinophil-associated disorder, cancer, asthma, inflammation, rheumatoid arthritis, allergic inflammation, inflammatory bowel disease, a gastrointestinal disorder, or fibrosis. In certain embodiments, the KIT-associated disorder is a fibrosis or inflammatory disorder, e.g., 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 disorder (e.g., a mast cell disease), a hematologic disorder, fibrosis (e.g., idiopathic pulmonary fibrosis (TPF), scleroderma, or myelofibrosis), or inflammation, such as asthma, rheumatoid arthritis, inflammatory bowel disease, or allergic inflammation.

In particular embodiments, the KIT-related disorder is a mast cell-related disorder. In particular embodiments, the KIT-associated disorder is an eosinophil-associated disorder, such as eosinophilic esophagitis (EoE).

Mast cells derived from myeloid progenitor cells are large cells present in connective tissue throughout the body, which are most abundant in submucosal tissue and dermis. They contain large particles that store a variety of mediator molecules (including vasoactive amine histamine) and have high affinity Fes receptors (FcsRI) that allow them to bind IgE monomers. IgE-binding antigens bound to mast cells trigger mast cell degranulation and mast cell activation, producing local or systemic tachyphylaxis. Mast cells therefore play an important role in inflammatory and allergic reactions. However, without proper balance and regulation, mast cells can also lead to adverse amplified responses to antigens observed in conditions such as allergy, idiosyncratic and rhinitis.

KIT signal transduction is important for mast cell development and stabilization, e.g., expansion of mast cells from their progenitor cells and their subsequent maturation and survival in the tissue in which they reside, homing of mast cells to their site of presence in vivo, and promotion of extracellular matrix protein adhesion by mast cells. Activating mutations in KIT, such as at amino acid residues 816 or 560 of KIT, have been associated with mast cell disease, characterized by overproduction of mast cells and gastrointestinal stromal tumors (GIST).

In particular embodiments, due to administration of a therapeutically effective amount of an anti-KIT antibody described herein, a method described herein for protecting, treating, or controlling a KIT-mediated disorder, e.g., fibrosis or inflammation (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: (i) Reduction or amelioration of fibrosis or inflammation (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation) and/or one or more symptoms associated therewith; (ii) A reduction in the duration of one or more symptoms associated with fibrosis or inflammation (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (iii) Prevention of fibrosis or recurrence of inflammation (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (iv) a reduction in hospitalization of the subject; (v) a reduction in length of hospitalization; (vi) Inhibition (e.g., partial inhibition) of the development of fibrosis or inflammation (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation) and/or one or more symptoms associated therewith; (vii) Enhancement or improvement of the therapeutic effect of another therapy (e.g., anti-inflammatory therapy, such as steroids); (viii) An increase in the number of patients in symptomatic relief (i.e., a period of time characterized by absence or minimal symptoms associated with inflammation); (ix) an increase in length of symptomatic relief in the patient; (x) reduced hospitalization rate; (xi) Reduced number of symptoms associated with fibrosis or inflammation (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); (xii) A decrease in 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 fibrosis or inflammation (e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, and allergic inflammation); and (xiii) improvement in quality of life as assessed by methods well known in the art, e.g., questionnaires.

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

As used herein, the term "a mast cell related disorder" or "a plurality of mast cell related disorders" refers to a disorder in which mast cell activity contributes to pathology and/or mast cells are present in abnormal amounts (e.g., greater than normal amounts or less than normal amounts) in a plurality of body parts. For example, a mast cell related disorder may exhibit accumulation of pathological mast cells in any or all of the organs and tissues that may be present and/or abnormal release of one or more mast cell mediators, such as inflammatory mediators. Non-limiting examples of inflammatory mediators released by mast cells include any of the following: (i) Particle-related mediators, including histamine, serotonin (5-hydroxytryptamine) and various proteases and peptidases; (ii) Eicosanoids such as prostaglandin D2 (PGD 2) and leukotriene C4 (LTC 4); 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 (TNFa) and chemokines including CCL-2, CCL-3, CCL-5 and CXCL8.

In a particular aspect, the mast cell-related disorder is a nervous system, e.g., a mast cell-related disorder of the central nervous system, such as NMO, NMOSD, MS or NF (e.g., NF type 1 (NF 1), NF type 2 (NF 2), or schwannoma).

MS is a chronic inflammatory demyelinating disorder of the central nervous system (brain and spinal cord) that includes the onset of white matter inflammation in the brain or spinal cord followed by damage to the individual's autoimmune system. These inflamed areas produce scars in the brain and spinal cord. The impairment disrupts the ability of portions of the nervous system to communicate, resulting in the development of a variety of symptoms, including physical, mental, and/or psychiatric problems. Forms of MS include, but are not limited to, recurrent forms, where symptoms occur as separate attacks, and progressive forms, where symptoms accumulate over time. Non-limiting examples of symptoms of MS include loss or alteration of sensory sensitivity, such as tingling, needle or paralysis, muscle weakness, very pronounced reflex, muscle cramp or movement difficulties, coordination and balancing difficulties (ataxia), language or swallowing problems, vision problems (nystagmus, optic neuritis or double vision), fatigue, acute or chronic pain, urination and defecation difficulties, mood problems, such as depression or mood instability, uhthoff phenomena (symptoms due to exposure to temperatures above common temperatures) and lmittee signs (specific feeling of electric shock along the back when bending the neck), and the therapeutic administration of antibodies or therapeutic fragments thereof, for example, to a human subject in need of such treatment, by specific amounts of the antibodies or therapeutic fragments thereof, are provided, by the therapeutic antibodies or the therapeutic fragments thereof, to the subject, have been described, in diagnostic guidelines for MS, 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. Symptoms obtainable from http:// www.ncbi.nlm.nih.gov/book/NBK 48919/. Symptoms of MS, such as autonomic nerves, vision, motor and sensory problems.

Other non-limiting examples of mast cell related disorders include, for example, allergies, atopic diseases, mast cell activation syndrome, allergic rhinitis, food and snake venom-related allergies (e.g., tree nuts, shellfish, fish, hymenoptera venom or bee sting allergies), psoriasis, atopic dermatitis, rosacea, eczema, tubular interstitial nephritis, glomerulonephritis, diabetic nephropathy, allograft rejection, amyloidosis, renal vascular ischemia, reflux nephropathy, polycystic kidney disease, drug-induced kidney disease, post-transplant ionic fibrosis and liver fibrosis (e.g., due to alcohol consumption, viral hepatitis b and c, and nonalcoholic steatohepatitis (NASH)), parasitic infections (e.g., schistosomiasis, amoeba, echinococcosis) and non-IgE mast cell mediated activation, such as angioedema and allergy.

Alternatively, the mast cell related disorder may be urticaria, in particular chronic urticaria, including chronic idiopathic urticaria, and chronic induced urticaria (i.e., chronic induced urticaria (CIndU)). In a specific embodiment, the mast cell related disorder is chronic induced urticaria. Chronic induced urticaria is a form of urticaria with its associated attributable causes of initiation, which often results in rubella (measles) or angioedema. In a specific embodiment, the chronic induced urticaria is cold urticaria (ColdU). When their skin is exposed to temperatures lower than the skin temperature, people suffering from cold urticaria experience symptoms such as itching, burning rashes and angioedema. In another specific embodiment, the chronic induced urticaria is symptomatic skin Scarification (SD). Symptomatic skin scarification is characterized by the appearance of rubella and flushing reactions that react to skin strokes, scratches or rubs, and it usually occurs within minutes of causing irritation. In another embodiment, the chronic inductive urticaria is cholinergic urticaria. Cholinergic urticaria is triggered by the body's sweating response to active or passive body heating, and is characterized by small (1-4 mm) rubella surrounded by bright red plaques (flares). Common causes include exercise, hot water bath/shower, fever, closing dressing, eating spicy food, and emotional stress. In another embodiment, the chronic inductive urticaria is a heat urticaria. In another embodiment, the chronic inductive urticaria is a delayed-type pressure urticaria. In another embodiment, the chronic inductive urticaria is solar urticaria. In another embodiment, the chronic inductive urticaria is vibratory urticaria. In another embodiment, the chronic inductive urticaria is contact urticaria. In another embodiment, the chronic inducible urticaria is waterborne. Antihistamines are approved therapies for chronic induced urticaria.

In various embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, fails one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard-of-care therapies for the disorder. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, fails antihistamine treatment for the disorder. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, fails to treat the disorder with H1-antihistamine. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, fails to treat H2-antihistamine for the disorder. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, fails to treat both H1-and H2-antihistamines for the disorder. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated for the disorder Treatment with one or more leukotriene receptor antagonists fails. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, fails to treat the disorder with one or more immunomodulatory or anti-inflammatory agents. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, uses an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab, for the disorderOr Li Geli bead mab (ligelizumab). In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, uses an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a degree of general Li Youshan antibody, for the disorder>Is a failure to treat. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, uses an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab (benralizumab), for the disorder >Is a failure to treat. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, fail treatment of the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab. In particular embodiments, a subject suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria) or eosinophiliaPatients with granulocyte-related disorders, such as eosinophilic esophagitis, fail treatment of the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte limumab (lirentelimab). In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, uses a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) Is a failure to treat. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, fail treatment of the disorder with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab). In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, fails treatment for the disorder with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has failed treatment with one or more Bruton's Tyrosine Kinase (BTK) inhibitors, e.g., lei Mibu brutinib (remibrutinib) and/or rizatrinib (rilzabrutinib), for the disorder. In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, fails to treat the disorder by: (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, e.g., omalizumab + >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), and/or (4) a treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, fails to treat the disorder by: (1) Antihistamine treatment (e.g., H1-and/or H2-antihistamine treatment), and (2) use of one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab- >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, e.g. anti-inflammatoryIL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (tezepelumab) (Tezspire ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, fails to treat the disorder by: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), and (3) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti- >Is a therapeutic agent. In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, fails to treat the disorder by: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), and (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumabOr Li Geli bead mab (ligelizumab). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, fails to treat the disorder by: (1) Using IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab +>Or Li Geli bead mab (ligelizumab), and (2) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent.

If the condition is refractory to the treatment, tolerates the treatment, relapses after the treatment and/or if the patient stops the treatment due to intolerance to the treatment, the patient is deemed to have failed treatment.

In various embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard-of-care therapies for the disorder. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to H1-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to H2-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, are refractory to both H1-and H2-antihistamine treatment. In certain embodiments, the mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related Disorders such as eosinophilic esophagitis are refractory to treatment with one or more leukotriene receptor antagonists. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, are refractory to treatment with one or more immunomodulatory or anti-inflammatory agents. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are useful for the use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumabOr Li Geli bead mab (ligelizumab) are refractory. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are treated with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupug Li Youshan anti-Is refractory to the treatment. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are treated with an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab (benralizumab) >Is refractory to the treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic-related disorders, such as eosinophilic esophagitis, are refractory to treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, for use with SiglTreatment with ec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), is refractory. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are treated with a TSLP or TSLPR inhibitor, such as an anti-TSLP or TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) Is refractory to the treatment. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophilic-related disorders, such as eosinophilic esophagitis, are refractory to treatment with C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab). In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophilic-related disorders, such as eosinophilic esophagitis, are refractory to treatment with CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophilic-related disorders, such as eosinophilic esophagitis, are refractory to treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizatrinib (rilzabrotinib). In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to the following treatments: (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, e.g., omalizumab + >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), and/or (4) a treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib). In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to the following treatments: (1) Antihistamine treatment (e.g., H1-and/or H2-antihistamine treatment), and (2) use of one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab- >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM )、Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738). In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to the following treatments: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), and (3) use of an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a Dupu Li Youshan antibody Is a therapeutic agent. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to the following treatments: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), and (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab). In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory to the following treatments: (1) Using IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumabOr Li Geli bead mab (ligelizumab), and (2) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent.

In various embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are resistant to one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard-of-care therapies for the disorder. In certain embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are resistant to antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are resistant to H1-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are resistant to H2-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are resistant to both H1-and H2-antihistamine treatment. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are resistant to treatment with one or more leukotriene receptor antagonists. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are resistant to treatment with one or more immunomodulatory or anti-inflammatory agents. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are resistant to the use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab). In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are tolerized with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dup Li Youshan anti-Duchesnea>Is a therapeutic agent. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are tolerized with an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab (benralizumab)>Is a therapeutic agent. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are resistant to treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are resistant to treatment with Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab). In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are tolerized with TSLP or TSLPR inhibitors, such as anti-TSLP or TSLPR antibodies, e.g., terfenarimab (Tezspire) ^TM ) Is a therapeutic agent. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are resistant to treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab). In particular embodiments, the fertilizerA large cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is resistant to treatment with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are resistant to treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizatrinib (rilzabrotinib). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis tolerance: (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, e.g., omalizumab + >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738), and/or (4) use of one or more BTK inhibitors, e.g., lei Mibu rutinib (remibrotinib) and/or rizutinibTreatment of nile (rilzabrotinib). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis tolerance: (1) Antihistamine treatment (e.g., H1-and/or H2-antihistamine treatment), and (2) use of one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab- >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis tolerance: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), and (3) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis tolerance: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), and (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +. >Or Li Geli bead mab (ligelizumab). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis tolerance: (1) Using IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab +>Or Li Geli bead mab (ligelizumab), and (2) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent.

In various embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and resistant to one or more prior treatments of the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard-of-care therapies for the disorder. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and resistant to antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophils Related conditions, such as eosinophilic esophagitis, are refractory and resistant to H1-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and resistant to H2-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and tolerant to both H1-and H2-antihistamine treatment. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with one or more leukotriene receptor antagonists. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with one or more immunomodulatory or anti-inflammatory agents. In particular embodiments, a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is paired with an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab Or Li Geli bead mab (ligelizumab) are refractory and tolerogenic. In specific embodiments, mast cell-related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are treated with an inhibitor of IL-4R, such as an anti-IL-4R antibody, e.g., dupup Li Youshan anti->Is refractory and tolerogenic. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are inhibited for use with IL-5RFormulations, e.g. anti-IL-5R antibodies, e.g. benralizumab>Is refractory and tolerogenic. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronic induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with Siglec8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab). In particular embodiments, a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is treated with a TSLP or TSLPR inhibitor, such as an anti-TSLP or TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) Is refractory and tolerogenic. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab). In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738. In certain embodiments, mast cell related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are refractory and resistant to treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizatrinib (rilzabrotinib). In certain embodiments, mast cell related disorders, such as nettleMeasles (e.g., chronic induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are refractory and resistant to the following treatments: (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, e.g., omalizumab + >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), and/or (4) a treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory and resistant to the following treatments: (1) Antihistamine treatment (e.g., H1-and/or H2-antihistamine treatment), and (2) use of one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab- >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory and resistant to the following treatments: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), and (3) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti- >Is a therapeutic agent. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory and resistant to the following treatments: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), and (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is refractory and resistant to the following treatments: (1) Using IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab +>Or Li Geli bead mab (ligelizumab), and (2) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent.

In various embodiments, a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after one or more prior treatments for the disorder. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard-of-care therapies for the disorder. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, are recurrent disorders that recur after H1-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, are recurrent disorders that recur after H2-antihistamine treatment. In particular embodiments, mast cell related disorders, such as urticaria (e.g., slow Induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are recurrent disorders that recur after H1-and H2-antihistamine treatment. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after treatment with one or more leukotriene receptor antagonists. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after treatment with one or more immunomodulatory or anti-inflammatory agents. In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are in use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumabOr Li Geli bead mab (ligelizumab). In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are in the use of an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dup Li Youshan anti-Duchesnea >Recurrent conditions that recur after treatment of (c). In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are treated with an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab (benralizumab)>Recurrent conditions that recur after treatment of (c). In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically inducedUrticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, are recurrent disorders that recur after treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab. In particular embodiments, mast cell-related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophilic-related disorders, such as eosinophilic esophagitis, are recurrent disorders that recur after treatment with Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte power mab (lirentelimab). In particular embodiments, mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, are treated with a TSLP or TSLPR inhibitor, such as an anti-TSLP or TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) Recurrent conditions that recur after treatment of (c). In particular embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab). In particular embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after treatment with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophilic-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizatrinib (rilzabrotinib). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) treatment with one or more leukotriene receptor antagonists (3) use of one or more immunomodulators or anti-inflammatory agents (e.g. IgE inhibitors, such as anti-IgE antibodies, e.g. omalizumab ++>Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g., IL-4R antibodies, e.g., dupu Li Youshan antibodyIL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumabIL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), and/or (4) a treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after: (1) Antihistamine treatment (e.g., H1-and/or H2-antihistamine treatment), and (2) use of one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab- >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>Inhibitors of IL-5R, e.g. anti-IL-5R antibodies, e.g. benralizumab (benralizumab)>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), and (3) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti- >Is a therapeutic agent. In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, is a recurrent disorder that recurs after: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), and (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab). In certain embodiments, a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilsEsophagitis is a recurrent condition that recurs after the following treatments: (1) Using IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab +>Or Li Geli bead mab (ligelizumab), and (2) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent.

In various embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has terminated one or more prior treatments for the disorder due to intolerance of the treatment. In certain embodiments, the one or more prior treatments include at least one standard of care therapy for the disorder. In certain embodiments, the one or more prior treatments are all standard-of-care therapies for the disorder. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has terminated antihistamine treatment for the disorder due to intolerance of the treatment. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has terminated treatment with H1-antihistamine for the disorder due to intolerance of the treatment. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has terminated treatment with H2-antihistamine for the disorder due to intolerance of the treatment. In particular embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, is treated Intolerance terminates H1-and H2-antihistaminic treatment for the condition. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has terminated treatment with one or more leukotriene receptor antagonists for the disorder due to intolerance of the treatment. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has terminated treatment with one or more immunomodulators or anti-inflammatory agents for the disorder due to intolerance of the treatment. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, have ceased use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab, for such disorders due to intolerance of treatmentOr Li Geli bead mab (ligelizumab). In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, have ceased use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dipivoxil Li Youshan, for such disorders due to intolerance of treatment >Is a therapeutic agent. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophil related disorders, such as eosinophilic esophagitis, have ceased use of an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab, for the disorder due to intolerance of treatment>Is a therapeutic agent. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, have terminated treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, for the disorder due to intolerance of the treatment. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, have terminated treatment with Siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), for the disorders due to intolerance of treatment. In particular embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, has been terminated with a TSLP or TSLPR inhibitor, such as an anti-TSLP or TSLPR antibody, for example, terlipzumab (Tezspire) ^TM ) Is a therapeutic agent. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, have terminated treatment with C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab), for the disorders due to intolerance of treatment. In particular embodiments, patients suffering from mast cell related disorders, such as urticaria (e.g., chronically induced urticaria) or eosinophilic related disorders, such as eosinophilic esophagitis, have terminated treatment with CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY3454738, for the disorders due to intolerance of the treatment. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, has ceased use of one or more BTK inhibitors for the disorder due to intolerance of the treatment, e.g., lei Mibu lutinib (remibrotinib) and/or rizatinib (rilzabr)utinib). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, has terminated the following treatment for the disorder due to intolerance of the treatment: (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, e.g., omalizumab + >Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), and/or (4) a treatment with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, has terminated the following treatment for the disorder due to intolerance of the treatment: (1) Antihistamine treatment (e.g., H1-and/or H2-antihistamine treatment), and (2) administration of one or more immunomodulatory or anti-inflammatory agents (e.g., igE inhibitors, Such as anti-IgE antibodies, e.g.omalizumab +.>Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumabIL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, has terminated the following treatment for the disorder due to intolerance of the treatment: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), and (3) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti- >Is a therapeutic agent. In certain embodiments, a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, ends up intolerance of treatmentThe following treatments for the disorder were stopped: (1) Antihistaminic therapy (e.g., H1-and/or H2-antihistaminic therapy), and (2) use of IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab). In certain embodiments, a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronic induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis, has terminated the following treatment for the disorder due to intolerance of the treatment: (1) Using IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab +>Or Li Geli bead mab (ligelizumab), and (2) use of IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan anti->Is a therapeutic agent.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who has symptomatic despite treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite the use of an H1-antihistamine alone or in combination with an H2-antihistamine and/or a leukotriene receptor antagonist for the disorder.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite treatment with one or more leukotriene receptor antagonists for the disorder.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, in spite of the administration of one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) for the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738), but still symptomatic. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitisThe patient, despite having performed the use of IgE inhibitors, such as anti-IgE antibodies, for the disorder, e.g. omalizumab +.>Or Li Geli bead mab (ligelizumab), but still symptomatic. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, in spite of the use of an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a dopp Li Youshan antibody, for said disorder>But still symptomatic. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, in spite of the use of an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab (benralizumab), for said disorder >But still symptomatic. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a subject suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorderSuch as eosinophilic esophagitis, which is symptomatic despite treatment with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, in spite of the use of a TSLP or TSLPR inhibitor, such as an anti-TSLP or TSLPR antibody, e.g., terstuzumab (tezspirare), for said disorder ^TM ) But still symptomatic. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite treatment with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, for the disorder.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite treatment with one or more BTK inhibitors, e.g., lei Mibu rutinib (remibrutinib) and/or rizabetinib (rilzabrutinib), for the disorder.

In one embodiment, the disclosure will be presented hereinThe anti-KIT antibody, antigen-binding fragment or conjugate thereof is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, who is symptomatic despite treatment with the following agents for the disorder: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, that is symptomatic despite treatment with the following agents for the disorder: (1) As described above, one or more antihistamines, and (2) as described above, one or more immunomodulators or anti-inflammatory agents. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, that is symptomatic despite treatment with the following agents for the disorder: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a subject suffering from a mast cell-related disorder, such as nettlePatients with rash (e.g., chronic induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, who are symptomatic despite treatment with the following agents for the disorder: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab). In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, that is symptomatic despite treatment with the following agents for the disorder: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab + >Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a Dupu Li Youshan antibody

In one embodiment, after treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails the treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment).

In one embodiment, after treatment with one or more leukotriene receptor antagonists for the disorder, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to treat with the one or more leukotriene receptor antagonists.

In one embodiment, one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) are used in the treatment of the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Following treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to the treatment with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, an IgE inhibitor, such as an anti-IgE antibody, e.g.omalizumab +. >Or LigeFollowing treatment with rituximab, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to treatment with the IgE inhibitor. In a specific embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g. a Dupu Li Youshan anti-I, is used in the treatment of said disorder>After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said patient fails said treatment with an IL-4R inhibitor. In specific embodiments, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g. benralizumab (benralizumab) is used for the disorder>After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said patient fails said treatment with an IL-5R inhibitor. In particular embodiments, after treatment of the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to treat the disorder with the IL-5 inhibitor. In particular embodiments, a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, is used in the disorder, e.g., li Lunte force mab (lire ntelimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails the treatment with the Siglec8 inhibitor. In particular embodiments, a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said patient fails said treatment with a TSLP or TSLPR inhibitor. In particular embodiments, after treatment of the disorder with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to treatment with the C5aR inhibitor. In particular embodiments, after treatment of the disorder with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails the treatment with the CD200R inhibitor.

In one embodiment, the anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment of the disorder with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib), wherein the patient fails the treatment with the one or more BTK inhibitors.

In one embodiment, following treatment for the condition with the following agents: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above, an anti-KIT antibody, antigen binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) as described above, one or more immunomodulators or anti-inflammatory agents, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to treat. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->Administration of an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein to a subject suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophilsA patient with a related disorder, such as eosinophilic esophagitis, wherein the patient fails the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails to treat. In one embodiment, following treatment for the condition with the following agents: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab + >Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient fails the treatment.

In one embodiment, after treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment with the one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment).

In one embodiment, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment of the disorder with one or more leukotriene receptor antagonists, wherein the disorder is refractory to the treatment with the one or more leukotriene receptor antagonists.

In one embodiment, one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) are used in the treatment of the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Following treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is for the treatment with one or more immunomodulators or anti-inflammatory agents Difficult to treat. In a specific embodiment, an IgE inhibitor, such as an anti-IgE antibody, e.g.omalizumab +.>Or Li Geli bead mab (ligelizumab), wherein the anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment with the IgE inhibitor. In a specific embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g. a Dupu Li Youshan anti-I, is used in the treatment of said disorder>After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said disorder is refractory to said treatment with an IL-4R inhibitor. In specific embodiments, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g. benralizumab (benralizumab) is used for the disorder >After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said disorder is refractory to said treatment with an IL-5R inhibitor. In particular embodiments, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a subject suffering from a mast cell-related disorder, such as urticaria (e.g., chronic), following treatment of the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiabInduced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, wherein said disorders are refractory to said treatment with an IL-5 inhibitor. In particular embodiments, after treatment of the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment with the Siglec 8 inhibitor. In particular embodiments, a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said disorder is refractory to said treatment with a TSLP or TSLPR inhibitor. In particular embodiments, after treatment of the disorder with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment with the C5aR inhibitor. In particular embodiments, after treatment of the disorder with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment with the CD200R inhibitor.

In one embodiment, the anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment of the disorder with one or more BTK inhibitors, e.g., lei Mibu lutinib and/or rizatrinib (rilzabrutinib), wherein the disorder is refractory to the treatment with the one or more BTK inhibitors.

In one embodiment, following treatment for the condition with the following agents: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above, an anti-KIT antibody, antigen binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) as described above, one or more immunomodulators or anti-inflammatory agents, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g.Dupu Li Youshan is resistant to->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab + >Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a Dupu Li Youshan antibodyThe anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory to the treatment. />

In one embodiment, after treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with the one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment).

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment with one or more leukotriene receptor antagonists for the disorder, wherein the disorder tolerates the treatment with the one or more leukotriene receptor antagonists.

In one embodiment, one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) are used in the treatment of the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Treatment of C5aR inhibitors, such as anti-C5 aR antibodies, e.g., ai Duoli mab (avdoralimab) and/or CD200R inhibitors, such as anti-CD 200R antibodies, e.g., LY 3454738)After treatment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, an IgE inhibitor, such as an anti-IgE antibody, e.g.omalizumab +. >Or Li Geli bead mab (ligelizumab), wherein the anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with the IgE inhibitor. In a specific embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g. a Dupu Li Youshan anti-I, is used in the treatment of said disorder>After treatment with an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with an IL-4R inhibitor. In specific embodiments, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g. benralizumab (benralizumab) is used for the disorder>After treatment of (a) an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to inhibition by the use of IL-5R Treatment of the preparation. In particular embodiments, after treatment with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with the IL-5 inhibitor. In particular embodiments, after treatment of the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with the Siglec 8 inhibitor. In particular embodiments, a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) After treatment with an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with a TSLP or TSLPR inhibitor. In particular embodiments, after treatment of the disorder with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with the C5aR inhibitor. In particular embodiments, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a subject suffering from a mast cell-related disorder, such as nettle, following treatment of the disorder with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738Patients with measles (e.g., chronic induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment with the CD200R inhibitor.

In one embodiment, the anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment of the disorder with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib), wherein the disorder is resistant to the treatment with the one or more BTK inhibitors.

In one embodiment, following treatment for the condition with the following agents: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above, an anti-KIT antibody, antigen binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) as described above, one or more immunomodulators or anti-inflammatory agents, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder tolerates the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder tolerates the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab + >Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->Administering an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is resistant to the disorderThe treatment is carried out.

In one embodiment, after treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and tolerable for the treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment).

In one embodiment, after treatment with one or more leukotriene receptor antagonists for the disorder, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the one or more leukotriene receptor antagonists.

In one embodiment, one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) are used in the treatment of the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., terbanpeiMonoclonal antibody (tezepelumab) (tezthread) ^TM ) Following treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and tolerable to the treatment with the one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, an IgE inhibitor, such as an anti-IgE antibody, e.g.omalizumab +. >Or Li Geli bead mab (ligelizumab), wherein the anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the IgE inhibitor. In a specific embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g. a Dupu Li Youshan anti-I, is used in the treatment of said disorder>After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, wherein said disorder is refractory and resistant to said treatment with an IL-4R inhibitor. In particular embodiments, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab, is used for the disorderAfter treatment of (a) an anti-KIT antibody as described herein, its useThe antigen binding fragments or conjugates are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the IL-5R inhibitor. In particular embodiments, after treatment of the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the IL-5 inhibitor. In particular embodiments, after treatment of the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the Siglec 8 inhibitor. In particular embodiments, a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) After treatment with an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the TSLP or TSLPR inhibitor. In particular embodiments, after treatment of the condition with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a subject suffering from a mast cell-related condition, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related condition, such as eosinophilic esophagitisA patient, wherein the disorder is refractory and resistant to the treatment with the C5aR inhibitor. In particular embodiments, after treatment of the disorder with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment with the CD200R inhibitor.

In one embodiment, the anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment of the disorder with one or more BTK inhibitors, e.g., lei Mibu lutinib and/or rizatrinib (rilzabrutinib), wherein the disorder is refractory and resistant to the treatment with the one or more BTK inhibitors.

In one embodiment, following treatment for the condition with the following agents: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above, an anti-KIT antibody, antigen binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) One or more antihistamines, as described above, and (2) one or more immunomodulators or anti-inflammatory agents, as described above, administering an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein to a subject suffering from a mast cell-related disorder, such as nettle Patients with a rash (e.g., chronic induced urticaria) or eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumabOr Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, e.g., an anti-IL-4R anti-Body, e.g. Dupu Li Youshan anti +.>The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is refractory and resistant to the treatment.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder, wherein the disorder is a recurring disorder that recurs following the treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment).

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment with one or more leukotriene receptor antagonists for the disorder, wherein the disorder is a recurring disorder that recurs following the treatment with one or more leukotriene receptor antagonists.

In one embodiment, one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) are used in the treatment of the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Following treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs following the treatment with one or more immunomodulatory or anti-inflammatory agents. In a specific embodiment, an IgE inhibitor, such as an anti-IgE antibody, e.g.omalizumab +. >Or Li Geli bead mab (ligelizumab), wherein the anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurring disorder that recurs following the treatment with the IgE inhibitor. In a specific embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g. a Dupu Li Youshan anti-I, is used in the treatment of said disorder>After treatment of (a) an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a subject suffering from a mast cell-related disorder, such as urticaria (e.g., chronically inducedUrticaria) or eosinophil-related disorders, such as eosinophilic esophagitis, wherein the disorder is a recurring disorder that recurs after the treatment with an IL-4R inhibitor. In specific embodiments, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g. benralizumab (benralizumab) is used for the disorder>The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurring disorder that recurs after the treatment with the IL-5R inhibitor. In particular embodiments, after treatment of the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs after the treatment with the IL-5 inhibitor. In particular embodiments, after treatment of the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte power mab (lirentelimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs after the treatment with a Siglec 8 inhibitor. In particular embodiments, a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) After treatment of a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to the patient in need thereofOr eosinophil-related disorders, such as eosinophilic esophagitis, wherein said disorder is a recurring disorder that recurs after said treatment with a TSLP or TSLPR inhibitor. In particular embodiments, after treatment of the disorder with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs after the treatment with the C5aR inhibitor. In particular embodiments, after treatment of a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs after the treatment with the CD200R inhibitor.

In one embodiment, the anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, following treatment of the disorder with one or more BTK inhibitors, e.g., lei Mibu lutinib and/or rizab (rilzabrutinib), wherein the disorder is a recurrent disorder that recurs following the treatment with one or more BTK inhibitors.

In one embodiment, following treatment for the condition with the following agents: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above, an anti-KIT antibody, antigen-binding fragment thereof, as described hereinThe fragments or conjugates are administered to a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurring disorder that recurs after the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) as described above, one or more immunomodulators or anti-inflammatory agents, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurring disorder that recurs after the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs after the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), binding an anti-KIT antibody described herein, antigen thereofThe fragment or conjugate is administered to a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurring disorder that recurs after the treatment. In one embodiment, following treatment for the condition with the following agents: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab + >Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the disorder is a recurrent disorder that recurs after the treatment.

In one embodiment, after treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) due to intolerance to the treatment.

In one embodiment, after treatment with one or more leukotriene receptor antagonists for the disorder, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the one or more leukotriene receptor antagonists due to intolerance to the treatment.

In one embodiment, one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab) are used in the treatment of the disorderOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) Following treatment with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient is terminated by intolerance to the treatment with one or more immunomodulators or anti-inflammatory agents. In a specific embodiment, an IgE inhibitor, such as an anti-IgE antibody, e.g.omalizumab +. >Or Li Geli bead mab (ligelizumab), administering an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein to a subject suffering from a mast cell-related disorderA patient suffering from a disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the IgE inhibitor due to intolerance to the treatment. In a specific embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g. a Dupu Li Youshan anti-I, is used in the treatment of said disorder>After treatment with an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the IL-4R inhibitor due to intolerance to the treatment. In specific embodiments, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g. benralizumab (benralizumab) is used for the disorder>After treatment with an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the IL-5R inhibitor due to intolerance to the treatment. In particular embodiments, after treatment of the disorder with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the IL-5 inhibitor due to intolerance to the treatment. In particular embodiments, the methods described herein will be followed by treatment of the disorder with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab) The anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the Siglec 8 inhibitor due to intolerance to the treatment. In particular embodiments, a TSLP or TSLPR inhibitor, such as an anti-TSLP or anti-TSLPR antibody, e.g., terfenarimab (Tezspire) ^TM ) After treatment with an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the TSLP or TSLPR inhibitor due to intolerance to the treatment. In particular embodiments, after treatment of the disorder with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the C5aR inhibitor due to intolerance to the treatment. In particular embodiments, after treatment of the disorder with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the CD200R inhibitor due to intolerance to the treatment.

In one embodiment, after treatment of the condition with one or more BTK inhibitors, e.g., lei Mibu lutinib (remibrotinib) and/or rizatrinib (rilzabrotinib), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related condition, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related condition, such as eosinophilic esophagitis, wherein the patient has terminated the treatment with the one or more BTK inhibitors due to intolerance to the treatment.

In one embodiment, following treatment for the condition with the following agents: (1) one or more antihistamines as described above, (2) one or more leukotriene receptor antagonists as described above, (3) one or more immunomodulators or anti-inflammatory agents as described above, and/or (4) one or more BTK inhibitors as described above, an anti-KIT antibody, antigen binding fragment or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment due to intolerance to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) as described above, one or more immunomodulators or anti-inflammatory agents, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate thereof described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment due to intolerance to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), and (3) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The antibodies described hereinThe KIT antibody, antigen-binding fragment or conjugate thereof is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment by intolerance to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) As described above, one or more antihistamines, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment due to intolerance to the treatment. In one embodiment, following treatment for the condition with the following agents: (1) IgE inhibitors, e.g. anti-IgE antibodies, e.g. omalizumab + >Or Li Geli bead mab (ligelizumab), and (2) an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., dupu Li Youshan antibody->The anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, wherein the patient has terminated the treatment by intolerance to the treatment.

Eosinophils are white blood cells activated by lymphocytes of the adaptive immune response and are important in defending against parasitic infections. The level of eosinophils in blood is generally low and can be significantly elevated under certain conditions, such as specific reactivity, which can lead to eosinophilia (an abnormally high eosinophil in blood).

As used herein, the term "eosinophil-related disorder" or "eosinophil-related disorders" refers to a disorder that occurs when eosinophils are present in a plurality of body parts in an abnormal amount (e.g., greater than normal or less than normal). For example, when the body produces excessive eosinophils, they can cause chronic inflammation, resulting in tissue damage. In certain aspects, eosinophil disorders may be associated with abnormal amounts of eosinophils in a tissue in response to an initiator for a period of time. For example, higher amounts of eosinophils may be produced in response to an initiator, such as an infection or allergen, but the larger amounts of eosinophils do not decrease at normal rates and therefore remain in larger amounts for longer periods of time than expected.

Eosinophil-related disorders may be diagnosed based on locations where eosinophil levels are elevated. Non-limiting examples of eosinophil-related disorders include allergic disorders, infectious disorders, blood disorders, immunological disorders and reactions, endocrine disorders, pulmonary conditions, gastrointestinal functional disorders, neurological disorders, rheumatic disorders, cardiac conditions, and kidney diseases. In a specific embodiment, the eosinophil-related disorder described herein is eosinophilic esophagitis (EoE). In certain aspects, eosinophilia is an eosinophil-related condition characterized by a peripheral blood eosinophil count greater than normal, e.g., greater than 450/. Mu.L. Eosinophilia may be induced or triggered by a variety of conditions, such as allergy or infection. In particular aspects, for example, elevated eosinophil levels are observed locally in the lung, heart, spinal cord or brain.

Non-limiting examples of neurological conditions involving eosinophils include central nervous system infections, ventricular and peritoneal bypass, and drug-induced adverse reactions. In certain aspects, an increase in eosinophil count or activity may be detected in cerebrospinal fluid (CSF) or in other samples of tissue derived from central nervous system fluid.

Non-limiting examples of eosinophil or mast cell related indications include upper airway disorders such as allergic rhinitis and sinusitis, foreign body inhalation, glottic stenosis, tracheal stenosis, laryngeal softening, vascular ring, chronic Obstructive Pulmonary Disease (COPD) and congestive heart failure, eosinophilic bronchitis, polychondritis, sarcoidosis, papillomatosis, arthritis (e.g., rheumatoid arthritis) and orbital necrotizing granulomatosis.

In certain embodiments, the "eosinophil-related disorder" or "eosinophil-related disorders" may include disorders in which eosinophil activity contributes to the disorder, e.g., disorders that occur when eosinophils are present in multiple body parts in an abnormal amount (e.g., greater than normal or less than normal).

In a certain aspect, according to the methods provided herein for treating eosinophil or mast cell-related disorders (e.g., eosinophil or mast cell-related disorders in the nervous system, e.g., the central nervous system), to a subject in need thereof at an administration dose and frequency that achieves one or more of the following in a subject diagnosed with an eosinophil or mast cell-related disorder: a decrease in eosinophil number and/or activity, a decrease in mast cell proliferation, a decrease in mast cell number or amount, an inhibition or decrease in mast cell activity, a decrease in mast cell-induced inflammatory factor production, a decrease in inflammatory factor production, a restoration of mast cell homeostasis, a decrease in mast cell migration, a decrease in mast cell adhesion, an inhibition or decrease in mast cell recruitment by eosinophils, and an antigen-mediated degranulation inhibition or decrease in mast cells.

In other embodiments, the KIT-associated disorder is a cancer, such as breast cancer, leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, mast cell leukemia), lung cancer (e.g., small cell lung cancer), neuroblastoma, gastrointestinal stromal tumor (GIST), melanoma, colorectal cancer, sarcoma (e.g., ewing's sarcoma), and germ cell tumor (e.g., seminoma). In particular embodiments, cancers treated or controlled or protected by the methods provided herein are characterized by a function-acquiring KIT mutation or overexpression of KIT.

In particular embodiments, the cancer treated according to the methods described herein may be any cancer type, including cancer or tumor cells expressing cell surface KIT or mutant forms thereof, which may be confirmed by any histological or cytological method known to those skilled in the art.

In certain embodiments, the cancer is metastatic. In certain embodiments, the cancer is an advanced cancer that has spread to the site of origin or outside the organ by local invasion or metastasis.

In particular embodiments, the cancer is a recurrent cancer that regrows at an initial or distal site after responding to the initial therapy (e.g., after surgery to remove the tumor and post-surgery adjuvant therapy). In some embodiments, the cancer is refractory cancer that develops even though an anti-tumor agent, such as a chemotherapeutic agent, is being administered or has been administered to the cancer patient. Non-limiting examples of refractory cancers are tyrosine kinase inhibitors, such as (imatinib mesylate), ->(SU 11248 or sunitinib), IRESSA ^TM (gefitinib), ->(erlotinib),(sorafenib) or VOTRIENT ^TM (pazopanib) refractory cancer. In some embodiments, the cancer is even by administering or having administered radiation therapy to a cancer patientChemotherapy, refractory cancer is still developing.

In particular embodiments, provided herein are methods for treating a refractory cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an antibody described herein, wherein the refractory cancer is resistant to an anti-cancer agent, such as a tyrosine kinase inhibitor (e.g.,(imatinib mesylate) or +.>(SU 11248 or sunitinib)) refractory or tolerant. Other non-limiting examples of tyrosine kinase inhibitors include 706 and aman 07 (nilotinib). RAD00I, PKC412, gefitinib (IRESSA) ^TM ) Erlotinib->Sorafenib (Sorafenib)Pazopanib (VOTRIENT) ^TM ) Axitinib, bosutinib, and cetirib(dasatinib), lapatinib +.>Ritamtinib, lenatinib, nilotinib +.>Semaxanib, tositunib, palldia ^TM ) Vandetanib (ZACTIMA) ^TM ) And varanib. In certain embodiments, the refractory cancer is initially treated with an anti-cancer agent, such as a tyrosine kinase inhibitor (e.g., a +_ >Or SU11248 (i.e. sunitinibNi), but resistance to anticancer agents has emerged. In certain embodiments, the subject has one or more mutations in KIT that confer tolerance to an anti-cancer agent, such as a tyrosine kinase inhibitor.

In particular embodiments, the antibodies described herein are administered to a patient who has previously received or is currently receiving one or more anti-cancer therapies, such as a chemotherapeutic agent or tyrosine kinase inhibitor (e.g.,(imatinib mesylate), ->(SU 11248 or sunitinib), IRESSA ^TM (gefitinib),(erlotinib), ->(sorafenib) or VOTRIENT ^TM (pazopanib)) or histone deacetylase inhibitors (e.g., vorinostat or suberoylanilide hydroxamic acid (SAHA)). In other embodiments, the antibodies described herein are administered to a patient who is or is suspected of being resistant or refractory to an anti-cancer therapy, e.g., a tyrosine kinase inhibitor, e.g., a ∈k->(imatinib mesylate),(SU 11248 or sunitinib), IRESSA ^TM (gefitinib), ->(erlotinib),(sorafenib) or VOTRIENT ^TM (pazopanib).

In particular embodiments, an antibody or antigen-binding fragment thereof (e.g., KIT-binding fragment thereof) or conjugate thereof described herein is administered to a patient who has previously received or is currently receiving one or more anti-cancer therapies, e.g., anti-growth factor receptor antibodies (e.g., anti-HER 2 antibodies, anti-EGFR antibodies, anti-VEGF antibodies, or anti-KIT antibodies) or anti-growth factor antibodies (e.g., anti-EGF antibodies, anti-VEGF antibodies). In other embodiments, the antibodies described herein are administered to a patient who is resistant or refractory to an anti-cancer therapy, e.g., an anti-growth factor receptor antibody (e.g., an anti-HER 2 antibody, an anti-EGFR antibody, an anti-VEGF antibody, or an anti-KIT antibody) or an anti-growth factor antibody (e.g., an anti-EGF antibody, an anti-VEGF antibody), or suspected of being resistant or refractory to an anti-cancer therapy.

In particular embodiments, as a result of administering a therapeutically effective amount of an anti-KIT antibody described herein, a method for protecting, treating, or controlling cancer in a subject in need thereof described herein can achieve at least one, two, three, four, or more of the following effects: (i) A reduction or improvement in the severity of cancer (e.g., leukemia, lung cancer, or gastrointestinal stromal cancer) and/or one or more symptoms associated therewith; a decrease in the duration of one or more symptoms associated with cancer (e.g., leukemia, lung cancer, or gastrointestinal stromal cancer); (iii) Prevention of recurrence of a tumor (e.g., a lung tumor or gastrointestinal stromal tumor); (iv) Regression of cancer (e.g., leukemia, lung cancer, or gastrointestinal stromal tumor) and/or one or more symptoms associated therewith; (v) a reduction in hospitalization of the subject; (vi) a reduction in hospitalization length; (vii) increased survival of the subject; (viii) Inhibition of the development of cancer (e.g., leukemia, lung cancer, or gastrointestinal stromal tumor) and/or one or more symptoms associated therewith; (ix) Enhancement or improvement of the therapeutic effect of another therapy (e.g., surgery, radiation therapy, chemotherapy or another tyrosine kinase inhibitor), (x) reduction or elimination of a cancer cell population (e.g., leukemia cell population, lung cancer cell population, gastrointestinal stromal tumor cell population), (xi) reduction of tumor or neoplasm growth, (xii) reduction of tumor size (e.g., volume or diameter), (xiii) reduction of the formation of newly formed tumor, (xiv) eradication, removal or control of primary, regional and/or metastatic cancer, (xv) eradication of tumor and/or edema-associated angiogenesis by reduction of tumor and/or edema-associated angiogenesis prior to surgery, (xvi) reduction of the number or size of metastases, (xvii) reduction of mortality, (xviii) increase of patient tumor-free survival, (xvix) increase of relapse-free survival, (xx) increase of patient number of remission, (xxi) reduction of hospitalization rate, (xxii) maintenance of tumor size and no increase, or increase of tumor less than after administration of standard therapy, such as by conventional methods available to those skilled in the art, such as Computed Tomography (CT), as measured by dynamic contrast enhanced MRI (DCE-MRI) or Positron Emission Tomography (PET) scanning; (xxiii) Prevention of the development or onset of one or more symptoms associated with cancer; (xxiv) an increase in the length of symptomatic relief of the patient; (xxv) a decrease in the number of symptoms associated with cancer; (xxvi) increased asymptomatic survival in cancer patients; (xxvii) A decrease in the concentration of one or more inflammatory mediators (e.g., cytokines or interleukins) in a biological sample (e.g., plasma, serum, cerebrospinal fluid, urine, or any other biological fluid) of a subject having a cancer (e.g., leukemia, lung cancer, or gastrointestinal stromal cancer); (xxviii) A reduction in Circulating Tumor Cells (CTCs) in the blood of a subject suffering from cancer (e.g., leukemia, lung cancer, or gastrointestinal stromal cancer); (xxix) Inhibition (e.g., partial inhibition) or reduction of tumor metabolism or perfusion; and (xxx) improvement in quality of life as assessed by methods well known in the art, e.g., questionnaires.

In certain aspects, provided herein are methods for killing cancer cells in an individual, wherein the methods comprise administering to an individual in need thereof an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or an antigen-binding fragment thereof or a conjugate thereof. In certain aspects, provided herein are methods for inhibiting growth or proliferation of cancer cells in an individual, wherein the methods comprise administering to an individual in need thereof an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or an antigen-binding fragment thereof or a conjugate thereof. In certain embodiments, a partial inhibition of growth or proliferation of cancer cells is achieved, e.g., at least about 20% to about 55% inhibition of growth or proliferation of cancer cells.

In certain aspects, provided herein are methods for reducing tumor size or burden in an individual in need thereof, wherein the methods comprise administering to the individual an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or antigen-binding fragment thereof or conjugate thereof.

In certain embodiments, an anti-KIT antibody described herein may be administered to a subject in need thereof by any suitable method. Non-limiting examples of methods of administration include mucosal, intradermal, intravenous, intratumoral, subcutaneous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. In one embodiment, the anti-KIT antibody or pharmaceutical composition thereof is administered systemically (e.g., parenterally) to a subject in need thereof. In another embodiment, an anti-KIT antibody or pharmaceutical composition thereof is administered topically (e.g., intratumorally) to a subject in need thereof. In particular embodiments, the anti-KIT antibody or pharmaceutical composition thereof is administered intravenously. In particular embodiments, the anti-KIT antibody or pharmaceutical composition thereof is administered subcutaneously. Each dose may or may not be administered by the same route of administration. In some embodiments, an anti-KIT antibody described herein may be administered simultaneously via multiple routes of administration or after the same or different other doses of anti-KIT antibody described herein.

When a disease or symptom thereof is being treated, administration of the substance typically occurs after the disease or symptom thereof has occurred. When a disease or symptom thereof is being prevented, administration of the substance typically occurs prior to onset of the disease or symptom thereof. In certain embodiments, an anti-KIT antibody described herein is administered to a subject prophylactically or therapeutically. The anti-KIT antibodies described herein can be administered to a subject prophylactically or therapeutically to prevent, reduce, or ameliorate a KIT-related disorder or disease (e.g., cancer, inflammation, fibrosis) or symptoms thereof.

According to the methods provided herein for treating a KIT-related disorder, eosinophil-related disorder, or mast cell-related disorder, the dose and frequency of administration of an anti-KIT antibody described herein, or a pharmaceutical composition thereof, to a subject (e.g., mammal, such as a human, dog, or cat) in need thereof will be effective while minimizing side effects. The exact dosage of an anti-KIT antibody or pharmaceutical composition thereof described herein to be administered to a particular subject may be determined based on factors related to the subject in need of treatment. Factors that may be considered include the severity of the disease state, the general health of the subject, the age and weight of the subject, diet, time and frequency of administration, combination with other therapeutic agents or drugs, response sensitivity, and tolerance/response to therapy. The dosage and frequency of administration of the anti-KIT antibodies or pharmaceutical compositions thereof described herein may be adjusted over time to provide adequate levels of anti-KIT antibodies or to maintain a desired effect.

The exact dosage to be used in the formulation will also depend on the route of administration and the severity of the condition or disease, and should be determined according to the judgment of the practitioner and each patient's circumstances.

In certain aspects, for an anti-KIT antibody described herein, the dose administered to a patient to prevent, protect, control, or treat a KIT-related disorder, eosinophil-related disorder, or mast cell-related disorder is typically from 0.1mg/kg to 100mg/kg of patient body weight. In particular embodiments, the dose administered to a patient to prevent, protect, control, or treat a KIT-related disorder, eosinophil-related disorder (e.g., eosinophilic esophagitis (EoE)), or mast-cell-related disorder (e.g., chronic urticaria, such as chronic induced urticaria) is about 3mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life in humans than antibodies derived from other species due to an immune response to foreign polypeptides. Thus, lower doses of human antibodies and less frequent administration are generally possible. Furthermore, the dosage and frequency of administration of the antibodies described herein can be reduced by modification, such as, for example, lipidation, to increase absorption and tissue penetration of the antibodies.

In one embodiment, about 0.001mg/kg (mg antibody per kg subject body weight) to about 500mg/kg of an anti-KIT antibody described herein is administered to prevent, protect, control or treat a KIT-related disorder, a mast cell-related disorder or an eosinophil-related disorder, such as eosinophilic esophagitis (EoE).

In some embodiments, an effective amount of an antibody provided herein is about 0.01mg to about 1,000mg. In particular embodiments, an "effective amount" or "therapeutically effective amount" of an anti-KIT antibody described herein refers to an amount of an anti-KIT antibody described herein sufficient to effect at least one, two, three, four, or more of the following effects: a reduction or improvement in the severity of a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder and/or one or more symptoms associated therewith; a decrease in the duration of one or more symptoms associated with a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder; prevention of recurrence of one or more symptoms of a KIT-related disorder, mast cell-related disorder, or eosinophil-related disorder and/or one or more symptoms associated therewith; reduced hospitalization of the subject; length of hospitalization is reduced; inhibition (e.g., partial inhibition) of development of a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder and/or one or more symptoms associated therewith; prevention of the development or onset of one or more symptoms associated with a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder; a decrease in the concentration of one or more inflammatory mediators (e.g., cytokines or interleukins) in a biological sample (e.g., plasma, serum, cerebrospinal fluid, urine, or any other biological fluid) of a subject having a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder; and improvement in quality of life as assessed by methods well known in the art, e.g., questionnaires. In some embodiments, an "effective amount" as used herein also refers to an amount of an antibody described herein that achieves the indicated result (e.g., inhibition of one or more KIT biological activities of a cell, such as inhibition of cell proliferation).

In some embodiments, an anti-KIT antibody described herein is administered as needed, e.g., weekly, biweekly (i.e., once every two weeks), monthly, bi-monthly, tri-monthly, etc.

In some embodiments, a single dose of one or more anti-KIT antibodies described herein is administered to a patient to hinder, prevent, protect, control, treat, and/or ameliorate a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder, such as eosinophilic esophagitis (EoE).

In particular embodiments, an anti-KIT antibody or pharmaceutical composition thereof is administered to a subject in accordance with the methods provided herein for treating a KIT-related disorder, a mast cell-related disorder, or an eosinophil-related disorder, wherein the anti-KIT antibody or pharmaceutical composition is administered for a period of time followed by a discontinuation of the administration of the anti-KIT antibody or pharmaceutical composition for a period of time (i.e., not administered for a period of time).

The methods provided herein comprise administering an anti-KIT antibody by any suitable route. Non-limiting examples of routes of administration include parenteral administration, e.g., subcutaneous, intramuscular, or intravenous administration, epidural administration, enteral administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration. The methods provided herein include an administration route that targets brain, ocular tissue or organ, spinal cord, or ear or auricular tissue. In particular aspects, the methods provided herein include an administration route that targets the nervous system, e.g., the central nervous system.

In particular embodiments, the methods provided herein comprise administering an anti-KIT antibody via a route suitable for crossing the blood brain barrier.

In addition, provided herein are combination therapies for treating a KIT-associated disorder or disease (e.g., a mast cell-associated disorder such as urticaria (e.g., chronically induced urticaria), eosinophil-associated disorders such as eosinophilic esophagitis, cancer, inflammation, fibrosis) comprising administering an anti-KIT antibody (e.g., a humanized anti-KIT antibody) or an antigen-binding fragment thereof (e.g., a KIT-binding fragment thereof) or an antibody conjugate thereof described herein to a subject in need thereof in combination with one or more other therapies (e.g., a second therapeutic agent such as a chemotherapeutic agent, tyrosine kinase inhibitor, PGP inhibitor, HSP-90 inhibitor, proteosome inhibitor, histone deacetylase inhibitor, antibody, or cytokine). In particular embodiments, provided herein are combination therapies for treating a KIT-associated disorder or disease (e.g., a mast cell-associated disorder, such as urticaria (e.g., chronically induced urticaria), eosinophil-associated disorder, such as eosinophilic esophagitis, cancer, inflammation, fibrosis) comprising administering an amount (e.g., a therapeutically effective or suboptimal amount) of an anti-KIT antibody described herein in combination with an amount (e.g., a therapeutically effective or suboptimal amount) of another therapy (e.g., a chemotherapeutic agent, tyrosine kinase inhibitor, antibody, cytokine, antihistamine, leukotriene receptor antagonist, immunomodulator, anti-inflammatory agent, or histone deacetylase inhibitor) to a subject in need thereof. In various embodiments, the combination therapy is administered to the subject substantially simultaneously, on the same day, on the same week, or on the same treatment cycle, or on similar or overlapping dosage administration schedules. In some embodiments, the combination therapy is administered to the subject simultaneously, concurrently or concomitantly. In other embodiments, the combination therapy is administered sequentially to the subject. In one embodiment, the anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to the subject prior to other therapies in the combination. In another embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to the subject following other therapies in the combination.

In combination therapy, one or more anti-KIT antibodies (e.g., humanized anti-KIT antibodies) provided herein or antigen-binding fragments thereof (e.g., KIT-binding fragments thereof) or antibody conjugates thereof may be administered prior to, concurrently with, or after administration of one or more other therapies (e.g., agents, surgery, or radiation therapy) for protecting, treating, controlling, and/or ameliorating a KIT-related disorder or disease (e.g., mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), eosinophil-related disorder, such as eosinophilic esophagitis, cancer, inflammation, fibrosis). The use of the term "combination" does not limit the order in which one or more anti-KIT antibodies and one or more other therapies are administered to a subject. In particular embodiments, the therapies may be administered sequentially or sequentially.

In particular embodiments, one or more other therapies, such as anti-cancer agents, for protecting, treating, controlling and/or ameliorating a KIT-associated disorder or disease (e.g., cancer, e.g., GIST, melanoma, or lung cancer), such as, for example, tyrosine kinase inhibitors (e.g., imatinib mesylate)Or Sunitinib (SUTENT)) or a histone deacetylase inhibitor (e.g., vorinostat or suberoylanilide hydroxamic acid (SAHA)), prior to, concurrently with, or subsequent to, administration of one or more anti-KIT antibodies (e.g., humanized anti-KIT antibodies) or antigen-binding fragments thereof (e.g., KIT-binding fragments thereof) provided herein, or antibody conjugates thereof.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, concurrently or concomitantly with one or more antihistamines (e.g., H1-and/or H2-antihistamine treatments) for said disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, and one or more antihistamines (e.g., H1-and/or H2-antihistamine treatment) for the disorder are sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with one or more leukotriene receptor antagonists for the disorder. At the same time, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, concurrently, or concomitantly with one or more leukotriene receptor antagonists for the disorder. At the same time, the anti-KIT antibodies, antigen-binding fragments or conjugates thereof described herein, and one or more leukotriene receptor antagonists for the disorder, are sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, the pharmaceutical composition is administered with one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +.>IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumab>IL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec 8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), in combination, administering an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, the pharmaceutical composition is administered with one or more immunomodulators or anti-inflammatory agents (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumabOr Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g., IL-4R antibodies, e.g., dupu Li Youshan antibody IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumabIL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), is administered simultaneously, concurrently or concomitantly with an anti-KIT antibody, antigen-binding fragment thereof or conjugate described herein to a subject suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or eosinophilsPatients with cell-related disorders, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with one or more immunomodulatory or anti-inflammatory agents for the disorder (e.g., igE inhibitors, such as anti-IgE antibodies, e.g., omalizumab>Or Li Geli bead mab (ligelizumab), IL-4R inhibitors, e.g. IL-4R antibodies, e.g. Dupu Li Youshan anti +. >IL-5R inhibitors, e.g. anti-IL-5R antibodies, e.g. benralizumabIL-5 inhibitors, such as anti-IL-5 antibodies, e.g., meperiab, siglec8 inhibitors, such as anti-Siglec 8 antibodies, e.g., li Lunte force mab (lirentelimab), TSLP or TSLPR inhibitors, such as anti-TSLP or anti-TSLPR antibodies, e.g., tertuzumab (Tezspirare) ^TM ) A C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab) and/or a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY 3454738), is sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, with an IgE inhibitor for said disorder, such as an anti-IgE antibody, e.g., omalizumabOr Li Geli bead mab (ligelizumab), to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, and for IgE inhibitors of such disorders, e.g. anti-IgE antibodies, e.g. omalizumab +.>Or Li Geli bead mab (ligelizumab), simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with an IgE inhibitor for the disorder, such as an anti-IgE antibody, e.g., omalizumab->Or Li Geli bead mab (ligelizumab) is sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a Dupu Li Youshan antibody, for use in the disorderIn combination, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a degree of general Li Youshan anti-I/O against a human or animal subject, for use in the disorder >Simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered to a subject suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic feedingPatients with tracheitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with an IL-4R inhibitor, such as an anti-IL-4R antibody, e.g., a dutch Li Youshan antibody, for use in the disorder>Sequentially to a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis.

In one embodiment, with an IL-5R inhibitor, such as an anti-IL-5R antibody, for use in the disorder, e.g., benralizumab (Benralizumab)In combination, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an IL-5R inhibitor, such as an anti-IL-5R antibody, e.g., benralizumab (bemalizumab), for use in said disorder is added to the composition >Simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with an IL-5R inhibitor, such as an anti-IL-5R antibody, for example, benralizumab (benralizumab), for use in the disorder>Sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or eosinophil-related disorderPatients suffering from symptoms such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, simultaneously, concurrently, or concomitantly with an IL-5 inhibitor, such as an anti-IL-5 antibody, e.g., meperiab, for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, and an IL-5 inhibitor for use in the disorder, such as an anti-IL-5 antibody, e.g., meperiab, are sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), for the disorder. In one embodiment, an anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, simultaneously, concurrently, or concomitantly with a Siglec 8 inhibitor, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), for use in the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein, and a Siglec 8 inhibitor for the disorder, such as an anti-Siglec 8 antibody, e.g., li Lunte force mab (lirentelimab), are sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-TSLP or TSLPR antibody, e.g., terfenarimab (tezspirare) ^TM ) In combination, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-TSLP or TSLPR antibody, e.g., terfenarimab (tezspirare) ^TM ) Simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with a TSLP or TSLPR inhibitor, such as an anti-TSLP or TSLPR antibody, for example, terlipzumab (tezthread ^TM ) Sequentially to a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), for use in the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, simultaneously, concurrently, or concomitantly with a C5aR inhibitor, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), for use in said disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, and a C5aR inhibitor for the disorder, such as an anti-C5 aR antibody, e.g., ai Duoli mab (avdoralimab), are sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, simultaneously, concurrently or concomitantly with a CD200R inhibitor, such as an anti-CD 200R antibody, e.g., LY3454738, for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein, and a CD200R inhibitor for the disorder, such as an anti-CD 200R antibody, e.g., LY3454738, are sequentially administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with one or more BTK inhibitors for the disorder, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib). In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, simultaneously, concurrently, or concomitantly with one or more BTK inhibitors for the disorder, e.g., lei Mibu lutinib and/or rizatrinib. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered sequentially to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, with one or more BTK inhibitors for the disorder, e.g., lei Mibu lutinib (remibrotinib) and/or rizibrutinib (rilzabrotinib).

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronically induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with (1) one or more antihistamines, as described above, (2) one or more leukotriene receptor antagonists, as described above, (3) one or more immunomodulators or anti-inflammatory agents, as described above, and/or (4) one or more BTK inhibitors, as described above, for use in the disorder. In one embodiment, an anti-KIT antibody, antigen binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, concurrently, or concomitantly with (1) one or more antihistamines, as described above, (2) one or more leukotriene receptor antagonists, as described above, (3) one or more immunomodulators or anti-inflammatory agents, as described above, and/or (4) one or more BTK inhibitors, as described above, for use in the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is sequentially administered with (1) one or more antihistamines, as described above, (2) one or more leukotriene receptor antagonists, as described above, (3) one or more immunomodulators or anti-inflammatory agents, as described above, and/or (4) one or more BTK inhibitors, as described above, for use in the disorder, to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis, in combination with (1) one or more antihistamines, as described above, and (2) one or more immunomodulators or anti-inflammatory agents, as described above, for the disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria), or an eosinophil-related disorder, such as eosinophilic esophagitis, concurrently or concomitantly with (1) one or more antihistamines, as described above, and (2) one or more immunomodulators or anti-inflammatory agents, as described above, for use in said disorder. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is administered sequentially with (1) one or more antihistamine as described above, and (2) one or more immunomodulator or anti-inflammatory agent as described above for the disorder to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis.

In one embodiment, as described above with (1) for the disorder,one or more antihistamines, (2) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumabOr Li Geli bead mab (ligelizumab) and (3) IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan antibody->In combination, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, with (1) one or more antihistamines as described above for the disorder, (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab>Or Li Geli bead mab (ligelizumab) and (3) IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan antibody->Simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with (1) one or more antihistamines, as described above, for the disorder, (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab @, as described above >Or Li Geli bead mab (ligelizumab) and (3) IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan antibody->Sequentially to a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis.

In one embodiment, with (1) one or more antihistamines as described above, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab, for use in the disorderOr Li Geli bead mab (ligelizumab), to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, with (1) one or more antihistamines as described above, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab +.>Or Li Geli bead mab (ligelizumab), simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with (1) one or more antihistamines, as described above, and (2) an IgE inhibitor, such as an anti-IgE antibody, e.g., omalizumab @, for use in the disorder >Or Li Geli bead mab (ligelizumab) sequentially to patients suffering from mast cell-related disorders, such as urticaria (e.g., chronic-induced urticaria) or eosinophil-related disorders, such as eosinophilic esophagitis。

In one embodiment, with (1) IgE inhibitors, such as anti-IgE antibodies, e.g., omalizumab, for said disordersOr Li Geli bead mab (ligelizumab) and (2) IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan antibody->In combination, the anti-KIT antibodies, antigen-binding fragments thereof, or conjugates described herein are administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, with (1) IgE inhibitors, such as anti-IgE antibodies, for said disorders, e.g. omalizumab +.>Or Li Geli bead mab (ligelizumab) and (2) IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan antibody->Simultaneously, concurrently or concomitantly, an anti-KIT antibody, antigen-binding fragment thereof, or conjugate described herein is administered to a patient suffering from a mast cell-related disorder, such as urticaria (e.g., chronic-induced urticaria) or an eosinophil-related disorder, such as eosinophilic esophagitis. In one embodiment, an anti-KIT antibody, antigen-binding fragment or conjugate thereof described herein is combined with (1) an IgE inhibitor for the disorder, such as an anti-IgE antibody, e.g., omalizumab +. >Or Li Geli bead mab (ligelizumab) and (2) IL-4R inhibitors, such as anti-IL-4R antibodies, e.g., dupu Li Youshan antibody->Sequentially to a patient suffering from a mast cell related disorder, such as urticaria (e.g., chronically induced urticaria) or an eosinophil related disorder, such as eosinophilic esophagitis.

In another embodiment, provided herein are combination therapies for treating a KIT-associated disorder or disease (e.g., a mast cell-associated disorder, such as urticaria (e.g., chronic-induced urticaria), eosinophil-associated disorder, such as eosinophilic esophagitis, cancer, inflammation, fibrosis) comprising administering to a subject in need thereof an amount of an anti-KIT antibody described herein (e.g., a humanized anti-KIT antibody) or an antigen-binding fragment thereof (e.g., a KIT-binding fragment thereof) or an antibody conjugate thereof in combination with an amount of another therapy (e.g., a chemotherapeutic agent, a tyrosine kinase inhibitor, or a histone deacetylase inhibitor). In particular embodiments, the combination therapy results in a synergistic effect. In certain embodiments, the combination therapy results in an additive effect.

In particular embodiments, provided herein are combination therapies for treating cancer comprising administering to a subject in need thereof an amount of an anti-KIT antibody described herein in combination with an amount of another therapy (e.g., surgery, radiation therapy, stem cell transplantation, or chemotherapy). In particular embodiments, the combination therapy results in a synergistic effect. In another embodiment, the combination therapy results in an additive effect.

In particular embodiments, provided herein are combination therapies for treating inflammation comprising administering to a subject in need thereof an amount of an anti-KIT antibody described herein in combination with an amount of another therapy (e.g., an anti-inflammatory therapy, e.g., a steroid therapy). In particular embodiments, the combination therapy results in a synergistic effect. In another embodiment, the combination therapy results in an additive effect.

Non-limiting examples of another therapy for combination with an antibody described herein include immunospecific binding toAnother anti-KIT antibody, one or more other antibodies (e.g., anti-HER 2 antibody, anti-EGFR antibody, anti-VEGF antibody), anti-inflammatory therapy, chemotherapy (e.g., microtubule disassembly blockers, antimetabolites, topoisomerase inhibitors, and DNA crosslinkers or damaging agents), radiation therapy, surgery, PGP inhibitors (e.g., cyclosporin A, verapamil), HSP-90 inhibitors (e.g., 17-AAG, STA-9090), proteosome inhibitors (e.g., bortezomib) and tyrosine kinase inhibitors (e.g., imatinib mesylate) Sunitinib (>Or SU 11248), gefitinib (IRESSA) ^TM ) Erlotinib->Sorafenib->Pazopanib (VOTRIENT) ^TM ) Axitinib, bosutinib, cetirib ∈>(dasatinib), lapatinibRitamtinib, lenatinib, nilotinib +.>Semaxanib, tositunib, palldia ^TM ) Vandetanib (ZACTIMA) ^TM ) And varanib). In a specific embodiment, another therapy for combination with an antibody described herein is imatinib mesylate.

Other non-limiting examples of another therapy for combination with an antibody described herein (e.g., a humanized anti-KIT antibody) or antigen-binding fragment thereof (e.g., KIT-binding fragment thereof) or an antibody conjugate thereof include histone deacetylase inhibitors, such as vorinostat or suberoylanilide hydroxamic acid (SAHA), or compounds having the formula (I), (II) or (III) as shown below. In particular embodiments, provided herein are methods of treating cancer (e.g., GIST or lung cancer), the methods comprising: (i) Administering an antibody described herein (e.g., a humanized anti-KIT antibody) or an antigen-binding fragment thereof (e.g., a KIT-binding fragment thereof) or an antibody conjugate thereof; and (II) a histone deacetylase inhibitor, e.g., vorinostat or suberoylanilide hydroxamic acid (SAHA) or a compound having the formula (I), (II) or (III) as shown below.

In one embodiment, provided herein are compounds of formula (I) for use in combination with an anti-KIT antibody in the methods described herein

Or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein

R ₁ Is a hydroxyamino group;

R ₂ and R is ₃ Each of which is independently the same as or different from each other, substituted or unsubstituted, branched or unbranched, and is hydrogen, hydroxy, alkyl, alkenyl, cycloalkyl, aryl, alkoxy, aryloxy, arylalkyloxy, or pyridine; or R is ₂ And R is ₃ Bonded together to form piperidine; and

n is an integer from 5 to 7.

In one embodiment, R ₂ Is a hydrogen atom and R ₃ Is a substituted or unsubstituted phenyl group. In certain embodiments, R ₃ Is methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2, 3-difluoro, 2, 4-difluoro, 2, 5-difluoro, 3, 4-difluoro, 3, 5-difluoro, 2, 6-difluoro, 1,2, 3-trifluoro, 2,3, 6-trifluoro, 2,4, 6-trifluoro, 3,4, 5-trifluoro, 2,3,5, 6-tetrafluoro, 2,34,5, 6-pentafluoro, azido, hexyl, tert-butyl, phenyl, carboxyl, hydroxyl, methoxy, phenoxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methoxycarbonyl, methylaminocarbonyl, dimethylamino-carbonyl or hydroxyaminocarbonyl-substituted phenyl. In another embodiment, R ₃ Is unsubstituted phenyl. In other embodiments, n is 6.

In one embodiment, provided herein are compounds of formula (II) for use in combination with an anti-KIT antibody in the methods described herein

Or a pharmaceutically acceptable salt or solvate thereof, wherein n is an integer from 5 to 8. In one embodiment, n is 6.

In one embodiment, provided herein is a compound (SAHA) of formula (III) for use in combination with an anti-KIT antibody in the methods described herein

Or a pharmaceutically acceptable salt, hydrate or solvate thereof.

The compounds of formulas I-III can be synthesized according to the methods described in the following patents: U.S. reissue patent No. re38,506 and U.S. patent No.6,087,367, each of which is incorporated herein by reference in its entirety.

In one embodiment, provided herein is a form I polymorph of SAHA for use in the methods described herein in combination with an anti-KIT antibody, characterized by an X-ray diffraction pattern substantially similar to that shown in fig. 13A of U.S. patent No.7,456,219, which is incorporated herein by reference in its entirety. In one embodiment, form I polymorph of SAHA is characterized by an X-ray diffraction pattern comprising characteristic peaks at about 9.0, 9.4, 17.5, 19.4, 20.0, 24.0, 24.4, 24.8, 25.0, 28.0 and 43.3 degrees 2θ as measured using a Siemens D500 automated powder diffractometer (range: 4-40 degrees 2θ; source: cu; λ=1.54 angstroms, 50kv,40 ma).

In certain embodiments, form I polymorph of SAHA is characterized by a Differential Scanning Calorimetry (DSC) thermogram having a single maximum at about 164.4±2.0 ℃ as measured by a Perkins Elmer DSC instrument with a heating rate of 10 ℃/min from 50 ℃ to at least 30 ℃ above the observed melting temperature.

Form I polymorphs of SAHA can be synthesized according to the methods described in U.S. patent No.7,456,219.

In one embodiment, provided herein is a crystalline composition comprising lysine and SAHA characterized by an X-ray diffraction pattern substantially similar to that shown in figure 1 of international patent application publication No. wo2008/042146, which is incorporated herein by reference in its entirety. In another embodiment, the crystalline composition is characterized by an X-ray diffraction pattern comprising characteristic peaks at about 6.8, 20.1, and 23.2 degrees 2-theta, as measured using a PANAnalytical X' Pert Pro X-ray powder diffractometer (range: 2-40 degrees 2-theta; source: cuK alpha 1 and K alpha 2). In another embodiment, the crystalline composition is characterized by an X-ray diffraction pattern comprising characteristic peaks at about 6.8, 12.6, 18.7, 20.1, 23.2, and 24.0 degrees 2-theta, as measured using a PANAnalytical X' Pert Pro X-ray powder diffractometer (range: 2-40 degrees 2-theta; source: cuK alpha 1 and K alpha 2). In another embodiment, the crystalline composition is characterized by an X-ray diffraction pattern comprising characteristic peaks at about 6.8, 12.0, 12.6, 16.4, 18.7, 20.1, 23.2, 24.0, 29.3 degrees 2 theta, as measured using a PANanalytical X' Pert Pro X-ray powder diffractometer (range: 2-40 degrees 2 theta; source: cukα1 and kα2).

In certain embodiments, the crystalline composition comprising lysine and SAHA is characterized by a Differential Scanning Calorimetry (DSC) thermogram wherein the endotherm of the crystalline composition exhibits an extrapolated onset temperature of about 182 ℃ as measured by a TA instrument Q1000 differential scanning calorimeter heated at a heating rate of 10 ℃/min from room temperature to 300 ℃.

Crystalline compositions comprising lysine and SAHA may be synthesized according to the methods described in international patent application publication No. wo 2008/042146.

In certain embodiments, the combination therapies described herein result in a synergy or synergistic effect. In particular embodiments, the synergy of the combination therapies allows for the use of lower doses (e.g., sub-optimal doses) of the anti-KIT antibodies and/or other therapies described herein and/or allows for less frequent administration of the anti-KIT antibodies or other therapies described herein to a subject. In certain embodiments, the ability to use lower doses of an anti-KIT antibody and/or other therapies and/or to administer an anti-KIT antibody or the other therapies less frequently reduces toxicity to a subject associated with administration of an anti-KIT antibody or the other therapies, respectively, while not reducing efficacy of an anti-KIT antibody or the other therapies in the treatment of KIT-related disorders or diseases, respectively. In some embodiments, the synergy results in an improvement in the efficacy of the anti-KIT antibodies described herein and/or the other therapies in treating a KIT-related disorder or disease. In some embodiments, the synergistic effect of the combination of an anti-KIT antibody described herein and one or more other therapies avoids or reduces adverse or undesirable side effects associated with the use of any monotherapy.

Provided herein are methods of inhibiting the activity of KIT in a cell expressing KIT, the method comprising contacting the cell with an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or an antigen-binding fragment thereof (e.g., a KIT-binding fragment thereof) or an antibody conjugate thereof. In particular embodiments, the methods inhibit KIT activity in a cell expressing KIT by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. Also provided herein are methods for causing or enhancing apoptosis in a cell expressing KIT, comprising contacting the cell with an effective amount of an antibody described herein. Also provided herein are methods for inducing or enhancing cell differentiation in a cell expressing KIT, the methods comprising contacting the cell with an effective amount of an antibody described herein.

KIT activity and, for example, the effect of antibodies on KIT activity can be routinely assessed using, for example, cell-based assays, such as those described herein.

Non-limiting examples of KIT activity that can be inhibited by the methods provided herein can include any KIT activity known or described in the art, e.g., KIT receptor dimerization, KIT receptor phosphorylation (tyrosine phosphorylation), KIT receptor (e.g., stat, AKT, MAPK or Ras signaling) downstream signaling, transcriptional regulation by KIT ligand (e.g., SCF) (e.g., SCF-induced transcriptional activation of c-Myc), cell proliferation, or induction or enhancement of cell survival.

In certain embodiments, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or antigen-binding fragment thereof (e.g., a KIT-binding fragment thereof) or an antibody conjugate thereof, sufficient to inhibit or antagonize KIT activity by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein and/or known to those of skill in the art (e.g., ELISA). In certain embodiments, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or an antigen binding fragment thereof (e.g., a KIT binding fragment thereof) or an antibody conjugate thereof, sufficient to inhibit or antagonize KIT activity by at least about 25%, 35%, 45%, 50%, 55%, or 65%, as assessed by methods described herein and/or known to those of skill in the art (e.g., ELISA). Non-limiting examples of KIT activity may include KIT receptor phosphorylation, KIT receptor signaling, KIT ligand (e.g., SCF) -mediated cell proliferation, KIT ligand (e.g., SCF) -mediated cell survival, and transcriptional activation of a KIT target gene (e.g., c-Myc).

In particular embodiments, a method for inhibiting KIT activity in a KIT-expressing cell comprises contacting the cell with an effective amount of an antibody described herein (e.g., a humanized anti-KIT antibody) or antigen-binding fragment thereof (e.g., KIT-binding fragment thereof) or an antibody conjugate thereof, sufficient to inhibit (e.g., partially inhibit) or antagonize downstream KIT signaling, e.g., signaling by a Src family kinase member, PI 3-kinase, or Ras-MAPK.

In another embodiment, a method for inhibiting (e.g., partially inhibiting) one or more KIT activities in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to inhibit or antagonize downstream KIT signaling, such as MAPK phosphorylation, AKT phosphorylation, or phosphorylation of Stat1, stat3, or Stat 5.

In certain embodiments, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to inhibit or reduce phosphorylation of AKT (e.g., phosphorylation of AKT by a KIT ligand (e.g., SCF)) by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein or known to one of skill in the art, e.g., western blot or ELISA assays or immunoblot assays. In certain embodiments, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, sufficient to inhibit or reduce phosphorylation of AKT (e.g., phosphorylation of AKT by a KIT ligand (e.g., SCF)) by at least about 25%, 35%, 45%, 55%, or 65%, as assessed by methods described herein or known to those of skill in the art, e.g., a western blot or ELISA assay or immunoblot assay.

In certain aspects, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT (e.g., a cancer cell) comprises contacting the cell with an effective amount of an antibody described herein sufficient to inhibit proliferation of the cell. Cell proliferation assays are described in the art and can be readily performed by those skilled in the art. For example, cell proliferation may be determined by measuring bromodeoxyuridine (BrdU) incorporation (see, e.g., hoshino et al, 1986,Int.J.Cancer 38,369;Campana et al, 1988, J.Immunol. Meth. 107:79) or (3H) thymidine incorporation (see, e.g., blechman et al, cell,1995,80:103-113; chen, J.,1996,Oncogene 13:1395-403; jelong, J.,1995,J.Biol.Chem.270:18367 73) by direct Cell counting at various time intervals (e.g., 12-hour or 24-hour intervals), or by detecting changes in transcription, translation or activity of known genes, such as oncogenes (e.g., fos, myc) or Cell cycle markers (Rb, cdc2, cyclin A, D, D2, D3, E, etc.). The levels and activity of such proteins and mRNAs can be determined by any method known in the art. For example, antibodies, including commercially available antibodies, can be used to quantify proteins by known immunodiagnostic methods such as ELISA, western blotting, or immunoprecipitation. mRNA can be quantified using methods well known and conventional in the art, for example, using Northern analysis, RNase protection, or polymerase chain reaction in combination with reverse transcription.

In particular embodiments, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT (e.g., a cancer cell) comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to inhibit cell proliferation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, as assessed by methods described herein or known to one of skill in the art (e.g., brdU incorporation assay). In particular embodiments, a method for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, sufficient to inhibit cell proliferation by at least about 25%, 35%, 45%, 55%, or 65%, as assessed by methods described herein or known to those of skill in the art (e.g., brdU incorporation assay). In particular embodiments, a method for inhibiting or antagonizing the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein sufficient to inhibit cell proliferation by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein or known to those of skill in the art (e.g., brdU incorporation assay).

In certain aspects, methods provided herein for inhibiting the activity of KIT in a cell expressing KIT (e.g., a cancer cell) comprise contacting the cell with an effective amount of an antibody described herein sufficient to reduce or inhibit survival of the cell. Cell viability assays are described in the art and can be readily performed by those skilled in the art. For example, cell viability may be assessed by using trypan blue staining or other cell death or viability markers known in the art. In particular embodiments, cellular ATP levels are measured to determine cell viability. In particular embodiments, cell viability may be measured over a period of three and seven days using standard assays in the art, such as CellTiter-Glo assay kit (Promega) that measures intracellular ATP levels. The reduction of cellular ATP is indicative of cytotoxic effects. In another embodiment, cell viability may be measured in a neutral red absorption assay. In other embodiments, visual observation of morphological changes may include enlargement, particle size, cells with rough edges, film appearance, rounding, detachment from the pore surface, or other changes. The following names are provided for these changes: t (100% toxicity), PVH (partial toxicity-heavier-80%), PH (partial toxicity-heavier-60%), P (partial toxicity-40%), ps (partial toxicity-lighter-20%) or 0 (non-toxic-0%), which corresponds to the observed degree of cytotoxicity. Regression analysis of these data determines 50% cytostatic (cytotoxic) concentrations (IC ₅₀ )。

In particular embodiments, a method provided herein for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to reduce or inhibit cell survival by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein or known to one of skill in the art (e.g., trypan blue dye exclusion assay). In particular embodiments, a method provided herein for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to reduce or inhibit cell survival by at least about 25%, 35%, 45%, 55%, or 65%, as assessed by methods described herein or known to those of skill in the art (e.g., trypan blue exclusion assay). In particular embodiments, a method provided herein for inhibiting the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein sufficient to reduce or inhibit cell survival by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein or known to those of skill in the art (e.g., trypan blue exclusion assay).

In particular embodiments, a method provided herein for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to cause apoptosis (i.e., programmed cell death). Methods for detecting apoptosis are described in the art and can be readily performed by one skilled in the art. For example, flow cytometry can be used to detect activated caspase 3, an enzyme that mediates apoptosis, or western blotting can be used to detect cleavage of poly (ADP-ribose) polymerase (PARP) in cells undergoing apoptosis (see, e.g., smolich et al, blood,2001, 97:1413-1421). Cleavage of PARP is an indication of apoptosis. In particular embodiments, a method provided herein for inhibiting or antagonizing the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein sufficient to induce or enhance apoptosis by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein or known to those of skill in the art (e.g., flow cytometry to detect activated caspase 3). In particular embodiments, a method provided herein for inhibiting or antagonizing the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein sufficient to induce or enhance apoptosis by at least about 25%, 35%, 45%, 55%, or 65%, as assessed by methods described herein or known to those of skill in the art (e.g., flow cytometry to detect activated caspase 3). In particular embodiments, a method provided herein for inhibiting the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein sufficient to induce or enhance apoptosis by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein or known to those of skill in the art (e.g., flow cytometry to detect activated caspase 3).

In particular embodiments, a method provided herein for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to cause differentiation. Methods for detecting differentiation are described in the art and can be readily performed by one skilled in the art. For example, flow cytometry may be used to detect the expression of one or more differentiation markers or the lack of expression of one or more undifferentiated markers in cells contacted with an antibody as described herein. Similarly, western blots may also be used to detect differentiation markers. Suitable differentiation markers and undifferentiated markers have been described and are one of the techniques in the art.

In particular embodiments, a method provided herein for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to induce differentiation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein or known to those of skill in the art (e.g., flow cytometry). In particular embodiments, a method provided herein for inhibiting (e.g., partially inhibiting) the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein, the contacting being sufficient to induce differentiation by at least about 25%, 35%, 45%, 55%, or 65%, as assessed by methods described herein or known to those of skill in the art (e.g., flow cytometry). In particular embodiments, a method provided herein for inhibiting the activity of KIT in a cell expressing KIT comprises contacting the cell with an effective amount of an antibody described herein sufficient to induce differentiation by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein or known to those of skill in the art (e.g., flow cytometry).

Non-limiting examples of cells that can be differentiated by the methods described herein include stem cells (e.g., embryonic stem cells, hematopoietic stem cells) and progenitor cells. Exemplary hematopoietic stem cell markers include CD38, CD34, CD59, CD133, sca-1, and ABCG2. Non-limiting examples of neural stem cell markers include nestin, PSA-NCAM, p75 neurotrophin R, and vimentin. Other non-limiting examples of stem cell markers include Oct4, sox2, klf4, LIN28, nanog, SSEA-3, SSEA-4, notch, and Wnt.

5.6 compositions

Provided herein are compositions, such as pharmaceutical compositions, comprising one or more anti-KIT antibodies (e.g., humanized antibodies) or antigen-binding fragments thereof described herein, or conjugates thereof described herein. In particular aspects, the compositions described herein may be used in vitro, in vivo, or ex vivo. In particular embodiments, provided herein are pharmaceutical compositions comprising an anti-KIT antibody (e.g., a humanized antibody) or antigen-binding fragment thereof described herein or a conjugate thereof described herein and a pharmaceutically acceptable carrier or excipient.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Antibodies of the desired purity may be prepared by combining the antibodies with an optional physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., easton, pa., remington: the Science and Practice of Pharmacy, 21 st edition (2006) Lippincott Williams)&Wilkins, baltimore, MD) are mixed to prepare therapeutic formulations provided herein containing one or more antibodies (e.g., humanized antibodies) in the form of a lyophilized formulation or an aqueous solution. At the dosages and concentrations employed, the acceptable carrier, excipient or stabilizer is non-toxic to the recipient and includes buffers such as phosphate, citrate and other organic acids; and/or nonionic surfactants, e.g. TWEEN ^TM 、PLURONICS ^TM Or polyethylene glycol (PEG).

Formulations, such as those described herein, may also contain more than one active compound (e.g., a molecule, e.g., one or more antibodies described herein) as necessary for the particular indication to be treated. In certain embodiments, the formulation comprises an antibody provided herein and one or more active compounds having complementary activities that do not adversely affect each other. These molecules are suitably present in combination in an amount effective for the intended purpose.

The formulation to be used for in vivo administration may be sterile. This is easily accomplished by filtration through, for example, a sterile filtration membrane.

In particular aspects, the pharmaceutical compositions provided herein contain a therapeutically effective amount of one or more anti-KIT antibodies (e.g., humanized antibodies) provided herein, and optionally one or more other prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. These pharmaceutical compositions are useful in preventing, protecting, treating, controlling or ameliorating a KIT-related disorder, an eosinophil-related disorder or a mast cell-related disorder, or one or more symptoms thereof.

Pharmaceutical carriers suitable for administration of the antibodies provided herein include any of these carriers known to those skilled in the art as suitable for the particular form of administration.

In addition, the antibodies described herein may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients (e.g., one or more other prophylactic or therapeutic agents).

The composition may contain one or more anti-KIT antibodies provided herein. In one embodiment, the antibodies are formulated in sterile solutions or suspensions for parenteral administration as suitable pharmaceutical preparations, such as solutions, suspensions, powders or elixirs. In one embodiment, the antibodies are formulated into pharmaceutical formulations suitable for oral administration, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, as well as transdermal patch formulations and dry powder inhalers.

In these compositions, one or more of the antibodies (or conjugates thereof) provided herein are admixed with a suitable pharmaceutical carrier. The concentration of the one or more antibodies in the composition may be effective, for example, to deliver an amount that treats, prevents, protects, or controls a KIT-related disorder, eosinophil-related disorder, or mast cell-related disorder, or one or more symptoms thereof, once administered.

In one embodiment, the composition is formulated for single dose administration. To formulate the composition, the weight fraction of the compound is dissolved, suspended, dispersed or otherwise admixed in a carrier of choice at an effective concentration such that the condition being treated is alleviated, prevented, or one or more symptoms are ameliorated.

In certain aspects, the antibodies (e.g., humanized antibodies) (or antibody-drug conjugates thereof) provided herein are included in a pharmaceutically acceptable carrier in an amount effective to exert a therapeutically useful effect without undesired side effects or with minimal or negligible undesired side effects on the patient being treated. The therapeutically effective concentration can be determined empirically by testing the compound in vitro and in vivo systems using conventional methods, and then extrapolated therefrom for the dosage of humans.

The concentration of antibody in the pharmaceutical composition will depend, for example, on the physicochemical characteristics of the antibody, the dosage schedule and the amount to be administered, as well as other factors known to those skilled in the art. In certain aspects, the concentration of antibody-drug conjugate in the pharmaceutical composition will depend, for example, on the physicochemical characteristics of the antibody and/or drug, the dosage schedule, and the amount to be administered, among other factors known to those of skill in the art.

In one embodiment, a therapeutically effective dose produces a serum concentration of antibody of about 0.1ng/ml to about 50-100 μg/ml. In another embodiment, the pharmaceutical composition provides a dose of about 0.001mg to about 2000mg of antibody per kilogram of body weight for administration over a period of time, e.g., daily, weekly, every 2 weeks, or every 3, 4, or 8 weeks. Pharmaceutical dosage unit forms can be prepared to provide from about 0.01mg to about 2000mg, and in one embodiment, from about 10mg to about 500mg of the combination of antibodies and/or other optional essential ingredients per dosage unit form.

In specific embodiments, the antibody-drug conjugates described herein are administered at an effective dose of about 1 to 100mg of antibody-drug conjugate per kilogram of body weight for a period of time, e.g., daily, weekly, every 2 weeks, or every 3 weeks.

The anti-KIT antibodies described herein may be administered at once, or may be divided into multiple smaller doses for administration at intervals. It will be appreciated that the exact dosage and duration of treatment will be related to the disease to be treated and may be determined empirically using known testing protocols or by extrapolation of in vivo or in vitro test data. It should be noted that the concentration and dosage values may also vary with the severity of the condition to be alleviated. It will also be appreciated that the particular dosing regimen may be adjusted over time for any particular subject according to individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Once the antibodies are mixed or added, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on several factors, including the intended form of administration and the solubility of the compound in the chosen carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the disease, disorder or condition to be treated and can be determined empirically.

The pharmaceutical compositions described herein are provided in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions, containing a suitable amount of a compound or a pharmaceutically acceptable derivative thereof for administration to humans and animals, such as mammals (e.g., cats or dogs). Pharmaceutical compositions are also provided in unit dosage forms, such as tablets, capsules, pills, powders, granules and oral solutions or suspensions, as well as oil-in-water emulsions, containing suitable amounts of the compound or a pharmaceutically acceptable derivative thereof for administration to humans and animals, such as mammals (e.g., cats or dogs). In one embodiment, the antibody is formulated and administered in unit dosage form or in multiple dosage form. A unit dosage form as used herein refers to physically separate units suitable as are known in the art for human and animal subjects and packaged separately. Each unit dose in combination with the desired pharmaceutical carrier, vehicle or diluent contains a predetermined amount of antibody sufficient to produce the desired therapeutic effect. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. The unit dosage form may be administered in portions or multiple times. Multiple dosage forms are multiple identical unit dosage forms packaged in a single container to be administered in separate unit dosage forms. Examples of multi-dose forms include vials, tablet or capsule bottles or pints or gallon bottles. Thus, a multi-dose form is a plurality of unit doses that are not separated in the package.

In certain embodiments, one or more anti-KIT antibodies described herein are in a liquid pharmaceutical formulation. Liquid pharmaceutically administrable compositions may be prepared, for example, by dissolving, dispersing or otherwise mixing an active compound as defined above and optionally a pharmaceutical adjuvant in a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, or the like, thereby forming a solution or suspension. The pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents, among others, if desired.

The actual methods of preparing these dosage forms are known or will be apparent to those skilled in the art; see, e.g., remington's Pharmaceutical Sciences (1990) Mack Publishing co., easton, PA; remington, the Science and Practice of Pharmacy, 21 st edition (2006) Lippincott Williams & Wilkins, baltimore, md.

Dosage forms or compositions containing antibodies in the range of 0.005% to 100% can be prepared with the remainder consisting of a nontoxic carrier. Methods for preparing these compositions are known to those skilled in the art.

In one embodiment, parenteral administration is also contemplated herein, characterized by subcutaneous, intramuscular, or intravenous injection. The injection may be prepared in conventional form as a liquid solution or suspension, as a solid form suitable for solution or suspension in a liquid prior to injection, or as an emulsion. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizing agents, and other such agents, if desired. Other routes of administration may include epidural, enteral, intracerebral, nasal, intraarterial, intracardiac, intraosseous infusion, intrathecal, and intraperitoneal administration.

Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products ready for incorporation with solvents just prior to use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready for incorporation with vehicles just prior to use, and sterile emulsions. The solution may be aqueous or non-aqueous.

If administered intravenously, suitable carriers include physiological saline or Phosphate Buffered Saline (PBS), and solutions containing thickening and solubilizing agents, such as dextrose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.

The pharmaceutical carrier further comprises ethanol, polyethylene glycol, and propylene glycol for a water miscible vehicle; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

Illustratively, intravenous or intra-arterial infusion of sterile aqueous solutions containing the active compound is an effective administration form. Another embodiment is a sterile aqueous or oily solution or suspension containing the active material for injection as needed to produce the desired pharmacological effect.

The anti-KIT antibodies described herein may be suspended in micronized or other suitable form. The form of the resulting mixture depends on several factors, including the intended form of administration and the solubility of the compound in the chosen carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the condition and can be determined empirically.

In other embodiments, the pharmaceutical formulation is a freeze-dried powder that can be reconstituted for administration as a solution, emulsion, and other mixture. They may also be reconstituted and formulated as a solid or gel.

Lyophilized powders are prepared by dissolving the antibodies provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art to be at about neutral pH in one embodiment. Subsequent sterile filtration of the solution and subsequent freeze-drying under standard conditions, known to those skilled in the art, provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder may be stored under suitable conditions, such as at about 4 ℃ to room temperature.

Reconstitution of such lyophilized powders with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. These amounts may be determined empirically.

The antibodies described herein can be formulated for topical or surface application, such as topical application to skin and mucous membranes, such as the eye, in the form of gels, creams and lotions, and for application to the eye or for intracisternal or intraspinal application. Topical administration for transdermal delivery, for ocular or mucosal administration, or for inhalation therapy is contemplated. Nasal solutions of the active compounds alone or in combination with other pharmaceutically acceptable excipients may also be administered.

Antibodies and other compositions provided herein can also be formulated to target specific tissues, receptors, or other areas of the subject's body to be treated. A variety of these targeting methods are well known to those skilled in the art. All such targeting methods for use in the compositions of the invention are contemplated herein. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. In some embodiments, an anti-KIT antibody described herein is targeted (or otherwise administered) to bone marrow. In some embodiments, an anti-KIT antibody described herein is targeted (or otherwise administered) to the gastrointestinal tract. In some embodiments, an anti-KIT antibody described herein is targeted (or otherwise administered) to the brain. In particular embodiments, the anti-KIT antibodies described herein are capable of crossing the blood brain barrier.

In particular embodiments, an anti-KIT antibody described herein is targeted (or otherwise administered) to an ocular tissue or organ. In particular aspects, compositions comprising an anti-KIT antibody described herein may be targeted to ocular tissues or organs as eye drops or gels. In particular aspects, compositions comprising an anti-KIT antibody described herein may be targeted to the ear.

Provided herein are pharmaceutical packages or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, as provided herein for one or more antibodies. Optionally, associated with such containers may be a notice in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency for human administration.

5.7 diagnostic methods

A label or otherwise detectable antibody that immunospecifically binds to a KIT antigen may be used for diagnostic purposes to detect, diagnose, or monitor a KIT-related disease.

Provided herein are methods for detecting KIT expression in a sample obtained from a patient suffering from a KIT-related disorder or disease. In particular embodiments, a method for detecting KIT expression in a sample obtained from a patient comprises contacting the sample with an anti-KIT antibody described herein and detecting the level of KIT expression in the sample, e.g., by correlating the binding of the anti-KIT antibody to KIT with the level of KIT expression. Detection methods are known to those skilled in the art.

In certain aspects, provided herein are methods for diagnosing a patient with a KIT-related disorder or disease. In a certain aspect, a method for diagnosing a subject with a KIT-related disorder or disease comprises contacting a cell or sample obtained from the subject with an anti-KIT antibody (or antigen-binding fragment thereof) described herein and detecting the expression level of KIT in the cell or sample. In certain embodiments, the method for diagnosing a patient with a KIT-related disorder or disease is an in vitro method. In particular embodiments, the method for diagnosing a patient with a KIT-related disorder or disease is an ex vivo method. In certain embodiments, the method further comprises administering an antibody or antigen binding fragment described herein to the patient after diagnosis.

In certain aspects, provided herein are methods of detecting a KIT-related disease comprising: (a) Determining expression of a KIT antigen in a cell or tissue sample of a subject using one or more antibodies described herein; and (b) comparing the level of KIT antigen to a control level, e.g., a level in a normal tissue sample (e.g., a sample from a patient not suffering from a KIT-related disorder, or from the same patient prior to onset), wherein an increase in the measured level of KIT antigen as compared to the control level of KIT antigen is indicative of the KIT-related disorder.

Detection methods are known to those skilled in the art. For example, the anti-KIT antibody may be conjugated to a detectable molecule (e.g., as described in section 5.1.1), and the detectable molecule may be visualized using standard techniques (e.g., microscopy). Antibodies as described herein can be used to determine KIT antigen levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (see, e.g., jalkanen et al, 1985, j. Cell. Biol.101:976-985; and Jalkanen et al, 1987, J.cell.biol.105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as ELISA and Radioimmunoassays (RIA). Suitable antibody assay tags are known in the art and include enzyme tags, such as glucose oxidase; radioisotopes, e.g. iodine @, of ¹²⁵ I、 ¹²¹ I) The carbon is ¹⁴ C) Sulfur ³⁵ S, tritium ³ H) The indium is ¹²¹ In) and technetium ⁹⁹ Tc); luminescent labels, such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin. In particular embodiments, the diagnostic methods described herein involve the use of naked or unlabeled antibodies that are not conjugated to a detectable marker, and the detection of the naked or unlabeled antibodies is indirect, e.g., by the use of a secondary antibody that can be labeled.

In certain embodiments, high expression of KIT in a sample relative to a normal control sample (e.g., a sample obtained from a healthy patient not suffering from a KIT-related disorder or disease) indicates that the patient suffers from a KIT-related disorder or disease.

A method for diagnosing a patient having a KIT-associated disorder or disease, such as cancer, in a sample obtained from the patient comprises contacting the sample with an anti-KIT antibody described herein and detecting the expression level of KIT in the sample. In certain embodiments, high expression of KIT in a sample relative to a normal control sample (e.g., a sample obtained from a healthy patient not suffering from a KIT-related disorder or disease) indicates that the patient suffers from a KIT-related disorder or disease.

In certain embodiments, the sample may be a tumor sample derived from a tumor of a patient or comprising tumor cells from a tumor of a patient. Examples of tumor samples herein include, but are not limited to, tumor biopsies, circulating tumor cells, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from a tumor or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples. In certain embodiments, the sample is a fixed tumor sample that has been histologically preserved using a fixative. In some embodiments, the sample is a formalin-fixed tumor sample that has been preserved using formaldehyde as a fixative. In certain embodiments, the sample is an embedded tumor sample surrounded by a hard and generally rigid vehicle, such as paraffin, wax, collodion, or resin. Embedding makes it possible to cut thin sections for microscopy or to create Tissue Microarrays (TMAs). In a specific embodiment, the sample is a paraffin-embedded tumor sample surrounded by a purified solid hydrocarbon mixture derived from petroleum. In certain embodiments, the sample is a frozen tumor sample, which is frozen or has been frozen. In particular embodiments, the sample, e.g., paraffin-embedded sample or frozen sample, is sectioned.

In certain aspects, a cancer or biological sample exhibiting KIT expression, amplification, or activation is a sample that expresses (including over-expresses) KIT receptors, has an amplified KIT gene, and/or otherwise exhibits KIT receptor activation or phosphorylation in a diagnostic test.

Also provided herein are detection and diagnosis of KIT-related diseases in humans. In one embodiment, the diagnosing includes: a) Administering (e.g., parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled antibody described herein to a subject; b) After administration, a waiting time interval to allow the labeled antibody to preferentially concentrate at sites in the subject expressing the KIT antigen (and clear unbound labeled molecule to background levels); c) Determining a background level; and d) detecting the labeled antibody in the subject, whereby detection of the labeled antibody above background levels indicates that the subject has a KIT-mediated disease. Background levels can be determined by a variety of methods, including comparing the amount of labeled molecules to be detected to standard values previously determined for a particular system.

It will be appreciated that the size of the subject and the imaging system used in the art will determine the amount of imaging portion required to produce a diagnostic image. In the case of radioisotope moieties, for human subjects, the amount of radioactivity injected will typically be in the range of about 5 to 20 millicuries ⁹⁹ Tc is in the range of Tc. The labeled antibody will then preferentially accumulate at the location of the cells containing the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their fragments "" (Tumor Imaging: the Radiochemical Detection of Cancer, S.W. Burchiel and B.A. Rhodes Main code, masson Publishing Inc. (1982) chapter 13).

Based on several variables, including the type of tag used and the form of administration, the time interval following administration to allow the labeled antibody to preferentially concentrate at sites in the subject and clear unbound labeled antibody to background levels is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

In one embodiment, monitoring of a KIT-mediated disease is performed by a method of repeatedly diagnosing the KIT-mediated disease, e.g., one month after the initial diagnosis, 6 months after the initial diagnosis, one year after the initial diagnosis, etc.

The presence of a marker molecule can be detected in a subject using methods known in the art for in vivo scanning. These methods depend on the type of label used. The skilled person will be able to determine the appropriate method for detecting a particular tag. Methods and apparatus that may be used in the diagnostic methods of the present invention include, but are not limited to, computed Tomography (CT), whole-body scanning, such as Positron Emission Tomography (PET), magnetic Resonance Imaging (MRI), and ultrasound scanning.

In particular embodiments, the molecules are labeled with a radioisotope and detected in a patient using a radiation responsive surgical instrument (Thurston et al, U.S. Pat. No.5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence-responsive scanner. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In another embodiment, the molecules are labeled with a paramagnetic label and detected in a patient using Magnetic Resonance Imaging (MRI).

6. Examples

The examples are provided herein by way of illustration and not by way of limitation.

6.1 example 1

Nucleic acid molecules encoding the Fc mutated H (heavy chain) and L (light chain) amino acid sequences of the anti-KIT antibodies (SEQ ID NO:23 and SEQ ID NO:24, respectively, see Table 6 below) were cloned directly into expression vectors. The construct was confirmed by sequencing and the vector was transfected into CHO cells. Stable transfection to establish cell lines expressing the antibodies is performed and subsequent drug selection is performed. The best expression line was selected based on IgG titers in the supernatant above background and amplified in the presence of drug selection and used at each stage The QKe system screens IgG expression.

Table 6: DNA sequences encoding the heavy and light chain sequences of an anti-KIT antibody.

After removal of the leader sequence, the resulting antibody was denoted antibody mAb1. The heavy and light chain amino acid sequences (after removal of the leader sequence) of the antibodies are shown in table 7 below.

Table 7: full length heavy and light chain amino acid sequences of mAb1.

The Fc domain of the heavy chain of the antibody comprises non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by EU numbering as set forth in Kabat. In the above sequence, the six non-naturally occurring amino acids are shown in bold, underlined text.

It was determined that mAb1 did not cause significant degranulation of FcgRI-expressing human mast cells compared to the corresponding antibody ("mAbc") with a wild-type (non-mutated) IgG1 Fc domain (as shown by the% release of β -hexosaminidase from human mast cells in culture). The release of β -hexosaminidase from human mast cells in culture (in the presence of ifnγ) was reduced by more than 50% by mAb1 compared to mAbc. In addition, mAb1 did not show significant Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation) compared to mAbc, even though crosslinked on THP-1 cells. Fc receptor-dependent KIT agonist activity (as determined by KIT phosphorylation using cross-linked Fc receptors) was reduced by more than 50% with mAb1 compared to mAbc.

6.2 example 2

Healthy volunteers were provided with a single infusion of either 0.3, 1, 3 or 9mg/kg mAb1 or placebo. Using detection of both alpha and beta forms of tryptaseTotal plasma tryptase levels were measured by assay. For each group, the tryptase values were normalized to 100% for the pretreatment values and to 0% for the lower assay limit (1 ng/mL). The mean and mean standard error are plotted against dose.

Figures 2A-2E show results demonstrating that mAb1 inhibits plasma tryptase in a dose-dependent manner.

6.3 example 3

A single dose of mAb1 resulted in an extended decrease in tryptase values below the quantitative level determined (1 ng/mL). Optionally, values below the detection level are plotted as 0.5 ng/mL. Absolute plasma tryptase values are shown.

Figures 3A and 3B show results demonstrating that a single dose of mAb1 provides durable tryptase inhibition at both 3mg/kg and 9 mg/kg.

6.4 example 4

Healthy volunteers were provided with a single infusion of either 0.3, 1, 3 or 9mg/kg mAb1 or placebo. The assay developed internally measures plasma levels of Stem Cell Factor (SCF), the unique ligand of the c-KIT/CD117 receptor, by using the Meso Scale Diagnostics (MSD) platform. SCF plasma levels rise in a dose-dependent manner, consistent with the allosteric blockade of KIT receptors by SCF. The mean and mean standard error are plotted against dose.

Figure 4 shows the results, which demonstrate that mAb1 induced a dose-dependent increase in plasma SCF levels.

6.5 example 5

M-07e cells were serum starved, then pretreated with i) mAb1, ii) a corresponding antibody with the same variable region sequence, but with unmutated (wild-type) human IgG1 sequence ("mAbc"), iii) isotype control antibody, or iv) a KIT-targeted small molecule kinase inhibitor (imatinib), and then stimulated with Stem Cell Factor (SCF). Phosphorylation was assessed by western blotting.

Fig. 5 shows the results from one experiment. The data shown represents 3 independent experiments. IgG = human IgG1 isotype control antibody; p-KIT = tyrosine phosphorylated KIT; p-AKT = phosphorylated AKT; p-ERK = phosphorylated ERK1/2. Overall, these results indicate that mAb1 is a more potent inhibitor of SCF-induced activation of wild-type KIT and downstream intracellular signaling pathways than the small molecule KIT inhibitors tested.

6.6 example 6

The effect of mAb1 and imatinib on SCF-dependent M-07e cell proliferation was also identified.

M-07e cells were serum starved, then pretreated with i) mAb1, ii) a corresponding antibody with the same variable region sequence, but with unmutated (wild-type) human IgG1 sequence ("mAbc") or iii) imatinib, and then stimulated with SCF. Cells were incubated at 37℃for 6 days.

The results are shown in fig. 6. The data represent 3 independent experiments and are expressed as the mean and mean standard error of three technical replicates.

Both mAb1 and mAbc showed similar dose-dependent inhibition of M-07e cell proliferation with average IC50 values (±sem; n=5; fig. 6) of 1.11±0.15nM and 1.12±0.22nM, respectively. By comparison, imatinib was less effective in inhibiting SCF-dependent M-07e cell proliferation with an average IC50 of 228±39nM (±sem) from 3 independent experiments. The antagonist activity of mAb1 was comparable to mAbc, indicating that introducing mutations into the Fc region of mAb1 did not affect its ability to inhibit KIT.

6.7 example 7

The binding affinity of mAb1 to recombinant human Fc-gamma receptor (FcrR) and human neonatal Fc receptor (FcRn) was identified and a balanced KD value was generated.

UsingQKe instrument binding to recombinant human Fc receptor was measured by biofilm interferometry. Histidine-tagged Fc receptors were captured on anti-pentahis biosensors and then exposed to serial dilutions of mAbc or mAb1 for 2-3 minutes followed by a dissociation step in the range of 5-10 minutes. Curve fitting was performed using instrument analysis software. Binding to FcRn was performed by a binding and dissociation step at pH 6.0 and pH 7.2.

The results are shown in fig. 7 and 8A-8N, which demonstrate that mAbc binds to fcri with high affinity (kd=3.39 nM), with medium affinity for fcriiia (kd=127 nM) and weak affinity for fcriia (kd=391 nM) and fcriiib (kd=816 nM). No binding to fcriib was observed. In contrast, mAb1 (concentration=500 nM) was detected to be unbound to any recombinant human FcrR.

Binding to human neonatal Fc receptor (FcRn) was tested at physiological pH 7.2 and at pH 6.0 mimicking endocytic conditions. At pH 7.2, no binding of mAbc to FcRn was observed, whereas mAb1 bound with moderate affinity (kd=77.1 nM). Mabc bound to FcRn with moderate affinity (kd=211 nM) at pH 6.0. However, mab1 bound to FcRn with significantly higher affinity and showed very slow dissociation (kd=0.38 nM) at pH 6.0.

These data indicate that Fc mutations in mAb1 abrogate FcrR interactions while enhancing in vitro interactions with FcRn.

6.8 example 8

Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of mAb1 was assessed using a commercially available ADCC Reporter Bioassay kit (Promega Corporation, madison, WI). ADCC Reporter Bioassay engineered Jurkat cells stably expressing the FcrRIIIa receptor V158 (high affinity) variant and nuclear factors driving the activated T cell (NFAT) response element expressed by firefly luciferase were used as effector cells. Binding of the fcriiia receptor on the surface of effector cells to the Fc effector portion of antibodies that bind to antigens on target cells results in cross-linking and activation of fcriiia signaling, which will cause expression of the NFAT reporter. To evaluate the ability of mAb1 and mAbc to induce ADCC, M-07e cells expressing KIT, transfected CHO cells (CHO-WT KIT) and human small cell lung cancer H526 cells were used as target cells. In addition, untransfected CHO cells that do not express KIT were used as control target cells. ADCC is considered to have been activated if a dose-dependent increase in the resulting reporter signal is observed.

As shown in fig. 9, mAbc elicited ADCC response to M-07e target cells expressing KIT, whereas mAb1 or isotype control did not. Similar results were observed with CHO-WT KIT and H526 cells. Overall, these data demonstrate that mAb1, unlike mAbc, does not elicit ADCC response against the examined KIT expressing target cells.

6.9 example 9

Fresh whole blood was incubated overnight with 40nM huIgG1 isotype control or 0.02, 0.2 or 40nM mAb1 in solution or in dry coating as indicated. Phytohemagglutinin (PHA) (10. Mu.g/mL) and Lipopolysaccharide (LPS) (10. Mu.g/mL) were used as positive controls. Plasma samples were harvested and stored frozen at-80 ℃. Cytokine levels were determined by using a multiplexed laser bead technique (Multiplexing Laser Bead Technology) implemented by Eve Technologies (Calgary, alberta, canada).

Fig. 10 shows the results, wherein the cytokine concentration of each donor is expressed as the average of two replicates. The mean cytokine concentration of all donors (n=6) was plotted using an error bar representing SEM. The results show that very little specific cytokine induction as a whole was observed by mAb1 relative to the human IgG1 isotype control.

6.10 example 10

Human patients are present and diagnosed with mast cell related diseases. mAb1 was administered intravenously to patients and monitored by clinical evaluation before, during and after response to treatment.

6.11 example 11 study of mAb1 in patients with chronic idiopathic urticaria

In this example a randomized, double-blind, placebo-controlled phase 1 multiple dose escalation study protocol is described to evaluate the safety, pharmacokinetics and pharmacodynamics of mAb1 as an additional therapy in patients with chronic idiopathic urticaria. The study type was interventional (clinical trial). Patient allocation is randomized. The intervention model is assigned in parallel. The mask type was double blind (participants and researchers).

The aim of the study was to investigate the safety, pharmacodynamics and pharmacokinetics of dose escalation of mAb1 in patients with chronic idiopathic urticaria, who are symptomatic despite treatment with antihistamines. There was up to a 2 week screening period, a 12 week double blind treatment period, and a 12 week post-treatment follow-up period. Patients received multiple doses of mAb1 or placebo as additional therapy for their antihistamines. Adult patients from 18 years to 75 years of age for all sexes are suitable for this study. Healthy volunteers were not received. It is estimated that the study will recruit 40 patients. As a group for this study, patients received intravenously administered mAb1 every 4-8 weeks; for the other groups of the study, patients received saline administered intravenously every 4-8 weeks.

Primary outcome measure:

1. safety was assessed by the incidence and severity of adverse events (time range: from day 1 (first dose) to day 169 (last follow-up)). The safety of multiple ascending doses of mAb1 was determined by drug related adverse events.

Secondary outcome measure:

1. pharmacokinetic assessment (time range: from day 1 (prior to first dose) to day 169 (last follow-up)). mAb1 serum concentrations were measured at the indicated follow-up.

2. Pharmacodynamic evaluation (time range: from day 1 (prior to the first dose) to day 169 (last follow-up)). Changes from baseline in urticaria activity score (UAS 7) in patients receiving mAb1 versus placebo.

3. Pharmacodynamic evaluation (time range: from day 1 (prior to the first dose) to day 169 (last follow-up)). Effect of mAb1 on tryptase and stem cytokine levels.

4. Pharmacodynamic evaluation (time range: from day 1 (prior to the first dose) to day 169 (last follow-up)). Changes from baseline in urticaria severity score (HSS 7) in patients receiving mAb1 versus placebo.

5. Pharmacodynamic evaluation (time range: from day 1 (first dose) to day 169 (last follow-up)). Changes from baseline in the itch severity score (ISS 7) in patients receiving mAb1 and placebo.

6. Safety evaluation (time frame: from day 1 (before dose administration) to day 169 (last follow-up)). Immunogenicity was assessed by measuring the development of anti-mAb 1 antibodies.

Key inclusion criteria:

1. men and women, 18-75 years old.

2. Diagnosis of chronic idiopathic urticaria (CSU) despite the use of H1-antihistamines alone or in combination with H2-antihistamines and/or leukotriene receptor antagonists, as defined below:

csu diagnosis >/=6 months.

b. Although H1-antihistamines are currently used, at any time prior to visit 1, itching and urticaria are present >/= for 6 consecutive weeks.

c. During the 7 days prior to treatment, UAS7 >/=16 and HSS7 >/=8.

d. At one of the screening follow-up shows, clinic UAS >/=4.

e. The H1-antihistamine is used alone or in combination with the H2-antihistamine and/or leukotriene receptor antagonist for at least 3 days before the study is entered and during the whole study.

3. Apart from CSU, there are no other obvious medical conditions that would pose additional risks or interfere with the study procedure.

4. Normal blood cell count and liver function test.

5. Men and women with a potential for pregnancy must agree to use very effective contraceptives during the study and 150 days after treatment.

6. An electronic diary of symptoms is willing and able to be completed daily during the study and follow the study follow-up schedule.

Key exclusion criteria:

1. pregnant or lactating women.

2. The cause of chronic urticaria is well-defined.

3. HIV, hepatitis b or hepatitis c infection is known.

4. Live vaccination was used within 4 weeks prior to study drug administration (subjects must agree to avoid vaccination during the study). The injection of inactivated vaccines, such as seasonal influenza, is allowed.

5. History of allergy.

There are other criteria for the treating physician to examine the candidate to confirm that it is appropriate for the study.

6.12 example 12-Single dose study of safety, pharmacokinetics and pharmacodynamics of mAb1 in patients with Cold contact urticaria, symptomatic skin scarification or cholinergic urticaria

Described herein are open label phase 1 single dose study protocols to evaluate the safety, pharmacokinetics, and pharmacodynamics of mAb1 as an adjunct therapy in patients with cold-contact urticaria, symptomatic skin scars, or cholinergic urticaria. The study type was interventional (clinical trial). The intervention mode is assigned to a single group.

The present study is an open-label phase 1 study that evaluates the safety, pharmacokinetics and pharmacodynamics of a single dose of mAb1 in patients with symptomatic cold-contact urticaria, symptomatic skin scars or cholinergic urticaria despite treatment with antihistamines. It is estimated that 10 patients with cold contact urticaria, 10 patients with symptomatic skin scars and 10 patients with cholinergic urticaria will be enrolled in three separate groups, totaling 30 patients. Prospective patients were screened by pre-recruited clinic testing and daily home diaries for 2 weeks. On day 1, a single dose of mAb1 was administered intravenously. Following treatment, the patient was followed for 12 weeks. Adult patients from 18 years to 75 years of age for all sexes are suitable for this study. Healthy volunteers were not received.

Primary outcome measure:

1. safety was assessed by the incidence and severity of adverse events (time frame: from day 1 to week 12). The safety of a single dose of mAb1 was determined by adverse events.

Secondary outcome measure:

1. for patients with cold-contact urticaria, the threshold temperature threshold (CTT) varies (time range: from day 1 to day 85). Such as by usingAs determined by the excitation test of (c), the critical temperature threshold varies over time from baseline.

2. For patients with symptomatic skin scarification, a threshold change (time range: from day 1 to day 85) was elicited. Such as by usingAs determined by the excitation test of (c), the excitation threshold varies over time from baseline.

3. For patients with cholinergic urticaria, the baseline urticaria activity score elicits a change (UASprovo) (time frame: from day 1 to day 85). Changes and percentages of responders from baseline as measured by uasprova.

4. Changes from baseline (time frame: from day 1 to day 85) in Urticaria Control Test (UCT). For UCT and modified UCT, the change and percentage of responders from baseline.

5. Blood biomarkers (time range: from day 1 to day 85). Blood samples before and after treatment were collected and analyzed for stem cell factor changes.

6. Blood biomarkers (time range: from day 1 to day 85). Blood samples were collected before and after treatment and analyzed for tryptase changes.

7. Pharmacokinetic assessment (time frame: from day 1 to day 85). mAb1 concentration was measured.

8. Immunogenicity evaluation (time frame: from day 1 to day 85). The patient is monitored for the presence of anti-drug antibodies.

Key inclusion criteria:

1. cold contact urticaria that is non-responsive to antihistaminesDiagnosis of measles, symptomatic skin scars or cholinergic urticaria. Diagnosing for more than or equal to 3 months; although antihistamines are used simultaneously, symptoms of both urticaria (rubella) and itching/burning/pain sensations. During screening, in the clinic, patients must have a positive cold stimulation test for cold contact urticaria; for symptomatic skin scarification, the patient must have a positiveAnd for cholinergic urticaria, the patient must have a positive pulse controlled muscle strength test (PCE) challenge test. The subject is at a stable antihistamine dose.

2. In addition to the diagnosis of cold contact urticaria, symptomatic skin scars, or cholinergic urticaria, no other risk factors will be introduced or other conditions will interfere with the study procedure as determined by the investigator based on the medical evaluation.

3. Male and female patients must use very effective contraception for at least 150 days from screening visit to receiving study treatment.

4. Willing and able to follow all study requirements and procedures, including completion of daily medication diaries and questionnaires.

Key exclusion criteria:

1. in addition to chronic urticaria, it is clear to diagnose urticaria or angioedema.

2. Prior biologic therapies (e.g., omalizumab, domino Li Youshan antibody, li Geli bead mab (ligelizumab)) were received within the last 3 months.

3. Treatment with immunosuppressants (e.g., systemic corticosteroids, cyclosporine, methotrexate, dapsone, cyclophosphamide, tacrolimus and mycophenolate mofetil, hydroxychloroquine, etc.) occurs over a 4 week or 5 half-life period.

4. Active covd-19 infection.

Infection with hiv, hepatitis b or hepatitis c.

6.13 example 13-mAb 1 reduces disease Activity and tryptase levels in patients with chronic induced urticaria

6.13.1 study background and summary

Chronic induced urticaria (CIndU) is characterized by Mast Cell (MC) -driven rubella that responds to cold in an initiator, such as cold in cold urticaria (cold) or skin scratch in symptomatic skin Scarification (SD). These diseases are often severe and debilitating, which can significantly affect patient longevity. MC require activation of their KIT receptors by stem cytokines to survive, proliferate and differentiate. MC loading is related to circulating tryptase (a protease secreted specifically by MC). mAb1 is a monoclonal anti-KIT antibody engineered to selectively inhibit Stem Cell Factor (SCF) -dependent KIT activation (see fig. 11). mAb1 demonstrated a significant dose-related reduction in circulating tryptase and was well tolerated overall in healthy volunteers. In this study, CIndU patients benefited from mAb1 treatment.

6.13.2 study design and method

In this ongoing open label phase 1b trial, coldU and SD patients refractory to antihistamine treatment received a single IV infusion of 3mg/kg mAb1 (as an additional treatment for H1-antihistamine) and were followed for 12 weeks. Patient symptoms were induced via a challenge test similar to the actual challenge. The main objective was to evaluate the safety/tolerability of mAb1 (adverse events and clinical laboratory tests). Secondary and research objectives include pharmacokinetic and pharmacodynamic assessments including changes in the threshold of stimulation from baseline, measurement of tryptase and stem cytokine levels, clinical outcome of activity (impact on urticaria symptoms, disease control, clinical response), quality of life assessment, and measurement of tissue mast cells by skin biopsy. Secondary goals included evaluating the effect of mAb1 on clinical activity and serum tryptase. Active endpoint includes a challenge test for disease severityPhysician global assessment (Phys-GA) and patient global assessment (Pat-GA). Excitation measurements are shown in FIGS. 12C-12D, 14A-14C, 15A-15D, and 16A-16D, respectivelyMean ± Standard Error (SE) of the test, biomarker and hematology. The number of skin MC assessed using non-diseased skin biopsies was counted by tryptase staining.

The study was modified to add patient groups with cholinergic urticaria.

The study was conducted substantially in accordance with the clinical trial protocol described in example 12.

6.13.3 study State

20 patients received study drug (i.e., mAb 1) at 3mg/kg by a single intravenous infusion and were included in the safety analysis. 11 had ColdU and 9 had SD. Patients have high disease activity as assessed by the challenge threshold test. In a ColdU patient, the baseline critical temperature threshold is 18.9deg.C/66F (range: 5-27deg.C/41-80.6F). In SD patients, baselineThe threshold was 3.8 (range: 3-4) for 4 needles. />

The 19 patients received full dose and were included in the activity analysis. 14 of the 19 patients completed a 12 week observation period; the 5-bit is in progress.

6.13.4 demographics and baseline disease characteristics

For identification of 20 patients, see table 8 below. All patients had prior antihistamine treatment.

Table 8: demographic and baseline disease characteristics

6.13.5 results of the study

As shown in fig. 12A-12D, a single dose of mAb1 (3 mg/kg) resulted in a rapid, pronounced and sustained response in cisdu patients refractory to antihistamines. In 95% (18/19) of patients (Col 100% (10/10) in dU patients (FIG. 12A) and 89% (8/9) in SD patients (FIG. 12B)) achieved Complete Response (CR). Prior to having a prior(omalizumab) all 3 patients (1 ColdU patient and 2 SD patients) experienced, including 2 +.>Complete response was observed in refractory patients. Rapid response onset and sustained persistence following dose administration were observed. By week 1 and week 4, respectively, most of the ColdU and SD patients experienced complete responses. In the cold u patients who completed the 12 week follow-up period (i.e., 8 cold u patients and 6 SD patients), CR was maintained for a median duration of 77 days, and in the SD patients for 57 days (fig. 12C and 12D). Of the 19 patients, 1 (SD patient) underwent Partial Response (PR). Cr=negative challenge test at ∈4 ℃ (for cold) or 0 needle (for SD). Pr=to 4 ℃ (for ColdU) or ≡2 (for SD).

The improved disease activity as assessed by Phys-GA and Pat-GA was consistent with the complete response as measured by the challenge test (FIGS. 13A and 13B, for ColdU and SD patients, respectively).

As shown in fig. 14A and 14B, a single 3mg/kg mAb1 dose resulted in rapid, significant and durable depletion of skin MC (87% depletion, see fig. 14A) and inhibition of serum tryptase (fig. 14B), as measured by biopsy. MC and tryptase kinetics showed similar trends over time (fig. 14C). In addition, skin MC number correlated positively with serum tryptase levels (fig. 14D).

The kinetics of skin MC and serum tryptase depletion reflect clinical activity. In particular, the kinetics of skin MC and serum tryptase depletion reflect a decrease in the challenge threshold (FIGS. 15A-15D). The data confirm that serum tryptase levels are robust pharmacodynamic biomarkers for assessing MC burden and clinical activity in CIndU patients and potentially other diseases with mast cell driven conditions.

In addition, mAb1 demonstrated good safety and tolerability. mAb1 is generally well tolerated in CIndU patients. The most common adverse events were changes in hair color (14/20 (70%)), infusion reactions (9/20 (45%)) and dysgeusia (8/20 (40%)). Changes in hair color (usually a small area of hair color lighten) and dysgeusia (usually a partial change in the ability to discriminate salty taste) are consistent with the inhibition of KIT signaling in other cell types and are expected to be fully reversible. Most adverse events were lighter. With longer observation period, the change in hair color was improved. Infusion reactions (usually manifested as urticaria and itching sensations) resolve spontaneously. A single severe infusion response of transient loss of consciousness occurred in patients with a history of syncope and was not due to MC activation, as measured by serum tryptase monitoring. The patient quickly recovers. Taste impairment is selective and transient. The hematology parameters generally remain within normal ranges (see fig. 16A-16D). A slight, transient and asymptomatic decrease in hemoglobin and White Blood Cell (WBC) parameters was noted (see fig. 16A-16D). However, there was no sign of clinically significant decrease in hematological parameters. This is an important finding for KIT inhibitors.

Overall, the data show that mAb1 demonstrates unprecedented MC depletion and a good safety profile, which provides significant potential as a cendu therapy for rapid, long lasting and meaningful alleviation, suggesting its potential to affect other diseases with mast cell involvement and providing an opportunity to evaluate MC involvement in a variety of diseases.

6.14 example 14-mAb 1 shows a rapid and sustained clinical response and improved quality of life in patients with chronic induced urticaria

6.14.1 introduction and goals:

chronic induced urticaria (CIndU) is a type of Mast Cell (MC) -driven disease characterized by itching-inducing rubella due to cold in known initiators, such as cold urticaria (cold u). MC surface receptor KIT is required for MC activation and survival. mAb1 is a monoclonal anti-KIT antibody that has shown significant inhibition of circulating tryptase (a marker of MC number) in the above studies in healthy adults.

6.14.2 material & method:

this example describes the information from CIndU: mid-term results of open label phase 1 test (NCT 04548869) for mAb1 in adults against histamine refractory ColdU or symptomatic skin Scarification (SD). Patients received a single Intravenous (IV) infusion of 3mg/kg mAb1 and were followed for 12 weeks. Safety results for 19 patients receiving study drug (ColdU: n=10, sd n=9) are described herein; clinical outcome results for 18 patients receiving full doses are described herein. By being in accordance with the method for ColdU Is based on the Critical Temperature Threshold (CTT) for SD>Is used for evaluating disease activity; the complete response was defined as a negative challenge test. Research evaluation also includes Urticaria Control Test (UCT) and dermatological quality of life index (DLQI).

6.14.3 results:

by the last evaluation (weeks 2-12), 17/18 patients achieved a complete response to the challenge test. In the ColdU patient, at baseline, the average CTT was 18.6deg.C (range: 5-27deg.C); the follow-up was 10 weeks after median treatment, and by the last evaluation (weeks 4-12), all 9 patients achieved complete response, with 6/9 patients achieving complete response at week 2. In SD patients, at baseline, the mean CFT was 3.8 (range: 3-4); the follow-up after median treatment was 7 weeks, 8/9 patients achieved complete response by the last evaluation (weeks 2-12), with 3/9 patients achieving complete response at week 2. All 7 (6 cold and 1 SD) patients who completed the 12 week evaluation maintained complete responses. The reduced firing threshold is accompanied by a durable inhibition of serum tryptase: in most patients, 4.4±1.6ng/ml from baseline to near or below the limit of detection at week 2 and maintained until week 8. Average UCT scores improved from 6 at baseline (range: 0-13) to 13 at week 4 (10-16), with 14 patients achieving UCT ≡12 (good control), with 5 achieving uct=16 by week 4 (complete control). For 15 patients, DLQI scores were available at baseline and week 4. Average DLQI scores improved from 11 at baseline (range: 2-21) to 2 at week 4 (range: 0-8) to 8 patients at week 4 achieved a score of 0 or 1 (disease had no impact on quality of life). The most common adverse events were changes in hair color in 11/19, transfusion reactions in 9/19 and dysgeusia in 8/19, most of which were mild.

6.14.4 conclusion:

mAb1 at a single 3mg/kg dose showed rapid and sustained clinical response and tryptase inhibition, improved quality of life, and was well tolerated overall. These data support KIT-driven MC inhibition as a potential therapeutic approach to control CIndU.

6.15 example 15-mAb 1 shows a rapid and sustained clinical response and improved quality of life in patients with chronic induced urticaria

6.15.1 study background and summary

Chronic induced urticaria (CIndU) is a Mast Cell (MC) driven disease characterized by itching and rubella caused by cold in cold urticaria (cold u) or skin scratching in symptomatic skin Scars (SD). Activation of KIT receptors by stem cytokines is essential for survival, proliferation and differentiation of MC. mAb1 is a monoclonal anti-KIT antibody engineered to selectively inhibit Stem Cell Factor (SCF) -dependent KIT activation (see fig. 11). In healthy volunteers, mAb1 caused a significant dose-dependent decrease in circulating tryptase (MC-loaded biomarker) and was well tolerated overall. As described in example 13, a single dose of mAb1 (3 mg/kg) was generally well tolerated and resulted in a rapid and sustained complete response in antihistamine refractory cendu (cold and SD) patients (negative challenge test). This example 15 describes other data from the same study. The data provided in this example 15 shows the effect of mAb1 on urticaria control and quality of life (QoL) in these patients.

6.15.2 study design and method

In this ongoing open label phase 1b trial, coldU and SD patients refractory to antihistamine treatmentA single IV infusion of 3mg/kg mAb1 was received and followed for 12 weeks. The main objective was to evaluate the safety/tolerability of mAb1 (adverse events and clinical laboratory tests). Secondary goals included evaluating the effect of mAb1 on clinical outcome and serum tryptase. Clinical efficacy assessment includes challenge testing Urticaria Control Test (UCT) and dermatological quality of life index (DLQI).

6.15.3 study State

21 patients received study drug (i.e., mAb 1) at 3mg/kg by a single intravenous infusion and were included in the safety analysis. 11 had ColdU and 10 had SD.

The 20 patients received full dose of study drug and were included in the UCT, DLQI and challenge test data.

20 of the 21 patients completed a 12 week observation period; bit 1 is in progress.

6.15.4 demographics and baseline characteristics

For identification of 21 patients, see table 9 below. All patients had prior antihistamine treatment.

Table 9: demographic and baseline characteristics.

6.15.5 results of the study

In the case of antihistamine refractory CIndU patients, a single dose of mAb1 (3 mg/kg) resulted in a rapid, pronounced and sustained response in 100% of patients, with 95% achieving a complete response, as described in example 13.

This notable response to the challenge test is also accompanied by significantly improved urticaria control and quality of life. A single 3mg/kg dose of mAb1 resulted in rapid and sustained improvement in urticaria control in cold and SD patients (fig. 17A, 17B, 18A and 18B). Rapid improvement in the UCT score was noted within 4 weeks and continued until week 12 (fig. 17A, 17B, 18A and 18B). By weeks 4 and 8, 80% and 100% of patients achieved a "well-controlled" state (UCT. Gtoreq.12), respectively (FIG. 18A). By week 8, 63% of patients achieved a "fully controlled" state (uct=16) (fig. 18B).

mAb1 also greatly reduced the disease impact on quality of life for both cold and SD patients (fig. 19A and 19B). By weeks 4 and 8, 93% and 92% of patients achieved ≡4-point reduction (minimal clinically significant differences (MCID)) in DLQI, respectively (fig. 20A). By weeks 4 and 8, 58% and 68% of patients achieved DLQI scores of 0-1 (disease had no effect on quality of life), respectively (fig. 20B).

In addition, as described above, a rapid and durable improvement in the challenge test was achieved with a 95% complete response (fig. 21A). A rapid, durable and significant tryptase reduction was also achieved (fig. 21B). The rapid and durable improvement in the excitation response reflects the decrease in tryptase (fig. 21A and 21B).

mAb1 is generally well tolerated in CIndU (cold and SD) patients. The most common adverse events were changes in hair color (15/21 (71%)), infusion reactions (9/21 (43%)) and dysgeusia (8/21 (38%)). Changes in hair color and dysgeusia are consistent with inhibition of KIT signaling in other cell types and are expected to be fully reversible. Most adverse events were lighter. With longer observation period, the change in hair color was improved. Infusion reactions (often manifested as urticaria and itching) are mostly mild and spontaneously resolved. A single severe infusion reaction occurs that is not attributable to MC activation. Taste impairment is selective and transient. The hematology parameters are typically maintained within normal ranges. A slight, transient and asymptomatic decrease in hemoglobin and White Blood Cell (WBC) parameters was noted. However, there was no sign of clinically significant decrease in hematological parameters.

Overall, the data show that mAb1 is generally safe and well tolerated, and that it has significant potential as a therapy for CIndU and other mast cell-related diseases.

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual patent publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

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35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> light chain variable region of anti-KIT antibody (VL 3)

<400> 15

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 16

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> light chain variable region of anti-KIT antibody (VL 4)

<400> 16

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> light chain variable region consensus sequences

<220>

<221> misc_feature

<222> (10)..(10)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having aromatic or aliphatic hydroxyl side chains; or in the alternative in a specific embodiment,

xaa is the amino acid Phe or Ser

<220>

<221> misc_feature

<222> (46)..(46)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having aliphatic or aliphatic hydroxyl side chains; or in the alternative in a specific embodiment,

Xaa is the amino acid Ala or Ser

<220>

<221> misc_feature

<222> (63)..(63)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

xaa is the amino acid Thr or Ser

<220>

<221> misc_feature

<222> (80)..(80)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

xaa is the amino acid Ser or Pro

<220>

<221> misc_feature

<222> (85)..(85)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having charged or acidic side chains; or in the alternative in a specific embodiment,

xaa is the amino acid Asp or Thr

<220>

<221> misc_feature

<222> (87)..(87)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having aromatic side chains; or in the alternative in a specific embodiment,

xaa is the amino acid Phe or Tyr

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Xaa Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Xaa Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Xaa

65 70 75 80

Glu Asp Phe Ala Xaa Tyr Xaa Cys Gln Gln Tyr Asn Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 18

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain variable region consensus sequences

<220>

<221> misc_feature

<222> (11)..(11)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having aliphatic side chains; or in the alternative in a specific embodiment,

Xaa is the amino acid Leu or Val

<220>

<221> misc_feature

<222> (20)..(20)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

xaa is the amino acid Leu or Val

<220>

<221> misc_feature

<222> (38)..(38)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having polar or basic side chains; or in the alternative in a specific embodiment,

xaa is amino acid Lys or Arg

<220>

<221> misc_feature

<222> (68)..(68)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

xaa is the amino acid Val or Ala

<220>

<221> misc_feature

<222> (70)..(70)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

xaa is the amino acid Leu or Ile

<220>

<221> misc_feature

<222> (73)..(73)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having acidic side chains; or in the alternative in a specific embodiment,

xaa is the amino acid Glu or Asp

<220>

<221> misc_feature

<222> (82)..(82)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having acidic or amide derivative side chains; or in the alternative in a specific embodiment,

xaa is the amino acid Gln or Glu

<220>

<221> misc_feature

<222> (91)..(91)

<223> Xaa can be any amino acid; or in a specific embodiment it may be

Amino acids having aliphatic hydroxyl side chains; or in the alternative in a specific embodiment,

xaa is the amino acid Ser or Thr

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Xaa Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Asn Trp Val Xaa Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Xaa Thr Xaa Thr Ala Xaa Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Xaa Leu Ser Ser Leu Arg Ser Glu Asp Xaa Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 19

<211> 464

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain sequence having mutation

<400> 19

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asp Tyr Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Phe Cys Ala Arg Gly Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

130 135 140

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

145 150 155 160

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

165 170 175

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

180 185 190

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

195 200 205

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

210 215 220

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

225 230 235 240

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Gln Gly Gly Pro

245 250 255

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr

260 265 270

Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

275 280 285

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Gln Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

340 345 350

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

370 375 380

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

405 410 415

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

420 425 430

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

435 440 445

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 20

<211> 234

<212> PRT

<213> artificial sequence

<220>

<223> light chain sequence

<400> 20

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn

35 40 45

Val Arg Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp

65 70 75 80

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn

100 105 110

Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 21

<211> 445

<212> PRT

<213> artificial sequence

<220>

<223> mAb1 full heavy chain sequence (including Fc)

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Val Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Gln Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Gln Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 22

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> mAb1 full-length light chain sequence

<400> 22

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 23

<211> 1392

<212> DNA

<213> artificial sequence

<220>

<223> DNA heavy chain sequence of anti-KIT antibody

<400> 23

atggagtggt cctgggtgtt cctgttcttt ctgtccgtga ccacaggcgt gcacagccag 60

gtgcagctgg tgcagtctgg agctgaggtg aagaagccag gagcttctgt gaagctgtcc 120

tgcaaggcca gcggctacac cttcacagac tactatatca actgggtgag acaggctcct 180

ggcaagggcc tggagtggat cgctcgcatc tatccaggct ctggcaacac ctactataat 240

gagaagttta agggccgggc caccctgaca gctgataaga gcacctctac agcttacatg 300

cagctgtcca gcctgagatc cgaggacacc gccgtgtact tctgcgctcg cggcgtgtac 360

tattttgatt attggggcca gggcaccaca gtgaccgtgt cttccgctag cacaaagggc 420

ccttccgtgt ttccactggc tcccagctct aagtccacca gcggaggaac agccgctctg 480

ggctgtctgg tgaaggacta tttcccagag cccgtgaccg tgagctggaa ctctggcgcc 540

ctgaccagcg gagtgcatac atttcctgct gtgctgcagt ccagcggcct gtactctctg 600

tcttccgtgg tgaccgtgcc aagctcttcc ctgggcaccc agacatatat ctgcaacgtg 660

aatcacaagc catccaatac aaaggtggac aagaaggtgg agcccaagag ctgtgataag 720

acccatacat gccccccttg tcctgctcca gaggctcagg gaggaccatc cgtgttcctg 780

tttccaccca agcctaagga caccctgtac atcacaaggg agccagaggt gacctgcgtg 840

gtggtggacg tgagccacga ggatcccgag gtgaagttca actggtacgt ggatggcgtg 900

gaggtgcata atgccaagac aaagccaagg gaggagcagt acaatagcac ctatcgggtg 960

gtgtctgtgc tgacagtgct gcaccaggac tggctgaacg gcaaggagta caagtgccag 1020

gtgtctaata aggccctgcc cgctcctatc gagaagacca tctccaaggc caagggccag 1080

cctagggagc cacaggtgta cacactgcct ccaagccggg acgagctgac caagaaccag 1140

gtgtctctga catgtctggt gaagggcttc tatccctctg atatcgctgt ggagtgggag 1200

tccaatggcc agcctgagaa caattacaag accacacccc ctgtgctgga ctccgatggc 1260

agcttctttc tgtattccaa gctgaccgtg gataagagca ggtggcagca gggcaacgtg 1320

ttttcttgtt ccgtgatgca tgaggctctg cacaatcatt acacacagaa gagcctgtct 1380

ctgtcccctg gc 1392

<210> 24

<211> 702

<212> DNA

<213> artificial sequence

<220>

<223> DNA light chain sequence of anti-KIT antibody

<400> 24

atgtccgtgc caacccaggt gctgggactg ctgctgctgt ggctgaccga cgccaggtgc 60

gatatcgtga tgacacagtc cccttccagc ctgtctgctt ccgtgggcga cagagtgacc 120

atcacctgta aggccagcca gaacgtgcgc accaatgtgg cttggtacca gcagaagcca 180

ggcaaggccc ccaaggctct gatctatagc gcctcttaca ggtatagcgg agtgcctgac 240

cggttcaccg gatccggaag cggaacagac ttcaccctga caatctcttc cctgcagcct 300

gaggacttcg ctgattactt ttgccagcag tacaactctt atccaaggac cttcggcggc 360

ggcacaaagg tggagatcaa gcggaccgtg gccgctccaa gcgtgttcat ctttccccct 420

tctgacgagc agctgaagtc tggcacagcc tccgtggtgt gcctgctgaa caacttctac 480

cccagagagg ccaaggtgca gtggaaggtg gataacgctc tgcagtctgg caattcccag 540

gagagcgtga ccgagcagga ctctaaggat tccacatata gcctgagctc taccctgaca 600

ctgtctaagg ccgattacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 660

ctgtccagcc cagtgacaaa gtccttcaat cgcggcgagt gt 702

<210> 25

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR1 of anti-KIT antibody (VH-CDR 1)

<400> 25

Gly Tyr Thr Phe Thr Asp Tyr

1 5

<210> 26

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR2 of anti-KIT antibody (VH-CDR 2)

<400> 26

Tyr Pro Gly Ser Gly Asn

1 5

<210> 27

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR3 of anti-KIT antibody (VH-CDR 3-AbM)

<400> 27

Gly Val Tyr Tyr Phe Asp Tyr Trp

1 5

<210> 28

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> light chain CDR1 of anti-KIT antibody (VL-CDR 1)

<400> 28

Ser Gln Asn Val Arg Thr Asn

1 5

<210> 29

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> light chain CDR2 of anti-KIT antibody (VL-CDR 2)

<400> 29

Ser Ala Ser

1

<210> 30

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> light chain CDR3 of anti-KIT antibody (VL-CDR 3)

<400> 30

Tyr Asn Ser Tyr Pro Arg

1 5

<210> 31

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR2 of anti-KIT antibody (VH-CDR 2)

<400> 31

Pro Gly Ser Gly

1

<210> 32

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR3 of anti-KIT antibody (VH-CDR 3)

<400> 32

Val Tyr Tyr Phe Asp Tyr

1 5

<210> 33

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR1 of anti-KIT antibody (VH-CDR 1-AbM)

<400> 33

Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile Asn

1 5 10

<210> 34

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR2 of anti-KIT antibody (VH-CDR 2-AbM)

<400> 34

Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr

1 5 10

<210> 35

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> light chain CDR1 of anti-KIT antibody (VL-CDR 1-Contact)

<400> 35

Arg Thr Asn Val Ala Trp Tyr

1 5

<210> 36

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> light chain CDR2 of anti-KIT antibody (VL-CDR 2-Contact)

<400> 36

Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr

1 5 10

<210> 37

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> light chain CDR3 of anti-KIT antibody (VL-CDR 3-Contact)

<400> 37

Gln Gln Tyr Asn Ser Tyr Pro Arg

1 5

<210> 38

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR1 of anti-KIT antibody (VH-CDR 1-Contact)

<400> 38

Thr Asp Tyr Tyr Ile Asn

1 5

<210> 39

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR2 of anti-KIT antibody (VH-CDR 2-Contact)

<400> 39

Trp Ile Ala Arg Ile Tyr Pro Gly Ser Gly Asn Thr Tyr

1 5 10

<210> 40

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain CDR3 of anti-KIT antibody (VH-CDR 3-Contact)

<400> 40

Ala Arg Gly Val Tyr Tyr Phe Asp Tyr

1 5

Claims

1. An antibody that immunospecifically binds to human KIT comprising:

(iii) A modified human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q and 322Q as numbered by EU numbering as set forth in Kabat.

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

3. The antibody of claim 1 or 2, wherein:

(i) The VL comprises the amino acid sequence:

DIVMTQSPSX _K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX _K2 LIYSAS YRYSGVPDRFX _K3 GSGSGTDFTLTISSLQX _K4 EDFAX _K5 YX _K6 CQQYNSYPRTFGGGTKVE IK (SEQ ID NO: 17), wherein X _K1 Is an amino acid having an aromatic or aliphatic hydroxyl side chain, X _K2 Is an amino acid having an aliphatic or aliphatic hydroxyl side chain, X _K3 Is an amino acid having 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 having a charged or acidic side chain and X _K6 Is an amino acid having an aromatic side chain; and is also provided with

(ii) The VH comprises the amino acid sequence:

4. The antibody of claim 3, wherein X _K1 Is amino acid F or S, X _K2 Is amino acid A or S, X _K3 Is the amino acid T or S, X _K4 Is the 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 the 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 Is amino acid S or T.

5. The antibody of any one of claims 1 to 4, wherein:

i) VL comprises SEQ ID NO: 13. 14, 15 or 16, and

ii) VH comprises SEQ ID NO: 8. 9, 10, 11 or 12.

6. The antibody of any one of claims 1 to 5, comprising:

(i) Comprising SEQ ID NO:14, VL of the amino acid sequence shown in seq id no;

(ii) Comprising SEQ ID NO:10, VH of the amino acid sequence shown in fig; and

7. The antibody of any one of claims 1 to 5, comprising:

(i) Comprising SEQ ID NO:14, VL of the amino acid sequence shown in seq id no;

(ii) Comprising SEQ ID NO:10, VH of the amino acid sequence shown in fig; and

(iii) A modified human IgG1 Fc region or domain comprising non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T and 256E as numbered by EU numbering as set forth in Kabat.

8. The antibody of any one of claims 1 to 5, comprising a heavy chain comprising the amino acid sequence:

9. the antibody of any one of claims 1 to 5, comprising a light chain comprising the amino acid sequence:

10. the antibody of any one of claims 1 to 5, comprising a heavy chain comprising the amino acid sequence:

11. A conjugate comprising the antibody of any one of claims 1 to 10 linked to a reagent.

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

13. A polynucleotide or combination of polynucleotides comprising nucleotide sequences encoding an antibody according to any one of claims 1 to 10 or VH and VL of said antibody.

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

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

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

17. A method for protecting, treating or controlling a KIT-related disorder comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to any one of claims 1 to 10, a conjugate according to claim 11, or a pharmaceutical composition according to claim 12.

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

19. The method of claim 18, wherein the KIT-related disorder is a mast cell-related disorder.

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

21. The method of claim 20, wherein the chronic urticaria is chronic-induced urticaria.

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

23. The method of claim 21, wherein the chronic-induced urticaria is symptomatic skin scarification.

24. The method of claim 21, wherein the chronically induced urticaria is cholinergic urticaria.

25. The method of claim 20, wherein the chronic urticaria is chronic idiopathic urticaria.

26. The method of claim 18, wherein the KIT-related disorder is an eosinophil-related disorder.

27. The method of any one of claims 17-26, further comprising administering a second therapeutic agent to the subject.

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

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

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

31. An in vitro method for diagnosing a subject with a KIT-related disorder, wherein the method comprises contacting a cell or sample obtained from the subject with an antibody according to any one of claims 1 to 10 and detecting the expression level of KIT in the cell or sample.

32. A method of producing an antibody, wherein the method comprises culturing the host cell of claim 15 and/or expressing the antibody using the host cell of claim 15.

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

34. A method for protecting, treating, or controlling chronic urticaria in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (1) an antibody that immunospecifically binds to human KIT or an antigen-binding fragment thereof, (2) a conjugate comprising the antibody or antigen-binding fragment thereof linked to an agent, or (3) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the conjugate, and a pharmaceutically acceptable carrier.

35. The method of claim 34, wherein the human KIT comprises SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

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

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

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

39. The method of any one of claims 34-38, wherein the antibody has reduced Fc receptor binding activity.

40. The method of claim 39, wherein the antibody has reduced FcrR binding activity.

41. The method of any one of claims 33 to 40, wherein the antibody does not cause significant degranulation of FcgRI-expressing human mast cells.

42. The method of any one of claims 33-41, wherein the antibody does not exhibit significant Fc receptor-dependent KIT agonist activity.

43. The method of any one of claims 33 to 42, wherein the antibody comprises:

44. The method of claim 43, wherein the chronic urticaria is chronic inducible urticaria.

45. The method of claim 44, wherein the chronically induced urticaria is cold urticaria.

46. The method of claim 44, wherein the chronic-induced urticaria is symptomatic skin scarification.

47. The method of claim 44, wherein the chronically induced urticaria is cholinergic urticaria.

48. The method of claim 43, wherein the chronic urticaria is chronic idiopathic urticaria.

49. The method of any one of claims 33 to 48, further comprising administering a second therapeutic agent to the subject.

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