Anti-kit antibodies and uses thereof

Abstract

To provide pharmaceutical compositions for treating or managing KIT-associated disorders comprising an antibody or antigen-binding fragment thereof that immunospecifically binds to a human KIT.SOLUTION: Provided are antibodies that immunospecifically bind to a human KIT antigen comprising the fourth and / or fifth extracellular Ig-like domains (that is, D4 and / or D5 domains), polynucleotides comprising nucleotide sequences encoding such antibodies, and expression vectors and host cells for producing such antibodies. The antibodies can inhibit KIT activity, such as ligand-induced receptor phosphorylation. Also provided herein are kits and pharmaceutical compositions comprising antibodies that specifically bind to a KIT antigen, as well as methods of treating or managing a KIT-associated disorder or disease and methods of diagnosing a KIT-associated disorder or disease using the antibodies.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application is filed under 35 U.S.C. §119(e) with the U.S. Provisional Application No. 100002664, each of which is incorporated herein by reference in its entirety. No. 61 / 675,751, filed July 25, 2012, incorporated herein by reference. The benefit of filed U.S. Provisional Application No. 61 / 675,762 is claimed.

[0002] This application is based on a file called "Sequence_List" created on July 18, 2013, which is 152,576 bytes in size. It was also submitted as an ASCII text file named "ting_12638-059-228.txt". The present invention includes a sequence listing, which is incorporated herein by reference in its entirety.

[0003] (1. Field) Provided herein are antibodies that specifically bind to KIT polypeptides, including antigen-binding Fragments, conjugates of such antibodies, polynucleotides encoding such antibodies Vectors and host cells for producing such antibodies, which immunospecifically bind to the KIT antigen and pharmaceutical compositions containing antibodies against KIT-associated disorders, their use for treating or managing KIT-associated disorders, and methods, as well as diagnostic methods. [Background technology]

[0004] (2.Background) KIT (or c-Kit) is a type III receptor tyrosine kinase encoded by the c-kit gene. KIT contains five extracellular immunoglobulin (Ig)-like domains, a single transmembrane domain, and inhibitory cell A vesicular juxtamembrane domain and a divided cytoplasm separated by a kinase insert segment kinase domain (see, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullric h 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 is and cloned as described in Yarden et al., EMBO J., 1987, 6:3341-3351. KIT is the receptor for stem cell factor ("SCF") ligand (also known as Steel factor or Kit ligand). It is also known as CD117 or stem cell factor receptor ("SCFR") because it is an SCF ligand. Binding of IL-1 to the first three extracellular Ig-like domains of KIT induces receptor dimerization, thereby It binds to the membrane via phosphorylation of specific tyrosine residues in the juxtamembrane and kinase domains. activating the tyrosine kinase activity of ATP (see, e.g., Weiss and Schlessinger, Cell, 1998, 94:277-280; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464. Members of the Stat, Src, ERK, and AKT signaling pathways mediate KIT signaling. It has been shown to be a downstream signaling factor for

[0005] The fourth (D4) and fifth (D5) extracellular Ig-like domains of KIT mediate receptor dimerization. (See, for example, International Patent Application Publication No. WO 2008 / 153926; Yuzawa et al., Cell, 2008). 007, 130:323-334).

[0006] KIT expression is found in mast cells, stem cells, brain cells, melanoblasts, ovarian cells, and cancer cells (e.g. It has been detected in various cell types, including leukemia cells. KIT is expressed in hematopoietic progenitor cells, mast cells, melanocytes, primordial germ cells, and Cajal It has been shown to be important for the normal growth of interstitial cells in the brain (see, e.g., Besmer, P., C Urr. Opin. Cell Biol., 1991, 3:939-946; Lyman et al., Blood, 1998, 91:1101-1134 Ashman, LK, Int. J. Biochem. Cell Biol., 1999, 31:1037-1051; Kitamura Mutat. Res., 2001, 477:165-171; Mol et al., J. Biol. Chem., 2003, 278 :31461-31464). Furthermore, KIT is involved in hematopoiesis, melanogenesis, and gametogenesis. (See Ueda et al., Blood, 2002, 99:3342-3349) .

[0007] Aberrant KIT activity has been implicated in several cancers, including SCF-independent KIT. Gain-of-function KIT mutations, which result in constitutive activation of KIT, are found in certain cancer cells and are associated with leukemia ( It has been associated with certain cancers, such as chronic myeloid leukemia (e.g., chronic myeloid leukemia) and gastrointestinal stromal tumors (e.g., See, for example, Mol et al., J. Biol. Chem., 2003, 278:31461-31464). Summary of the Invention

[0008] (3. Overview) Provided herein, in one embodiment, is an extracellular domain of KIT (e.g., human KIT). an antibody that immunospecifically binds to domain 4 (D4) (or the D4 region) of KIT and inhibits KIT activity; Binding fragments and conjugates thereof, as well as related compositions, reagents, and methods .

[0009] In one aspect, provided herein are antibodies that immunospecifically bind to D4 of human KIT. 1. A method for the preparation of a human antibody or antigen-binding fragment thereof, comprising: (i) VL CDs having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively a light chain variable region (“VL”) comprising R1, VL CDR2, and VL CDR3; and (ii) VH C having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. a heavy chain variable region ("VH") comprising DR1, VH CDR2, and VH CDR3 and wherein the antibody or antigen-binding fragment thereof comprises: In one embodiment, the VL and VH of the antibody or antigen-binding fragment thereof provided herein are In certain embodiments, the antibody is non-immunogenic in humans. It can be expressed in Chinese ovariectomy (CHO) cells at titers of at least 0.45 μg / mL In certain embodiments, the antibody is expressed in Chinese hamster ovary (CHO) cells. , at least 0.3 μg / mL, at least 0.6 μg / mL, at least 0.75 μg / mL, or at least It can be expressed at a titer of 1 μg / mL.

[0010] In some embodiments, provided herein are antibodies that immunospecifically bind to D4 of human KIT. An antibody, or antigen-binding fragment thereof, or a conjugate thereof, comprising: Amino acid sequence: [ka] (where X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, and X K2 teeth, an amino acid having an aliphatic side chain or an aliphatic hydroxyl side chain, X K3 is an aliphatic hydrochloride an amino acid with a xyl side chain, X K4 is an amino acid with an aliphatic hydroxyl side chain or P and X K5 is an amino acid with a charged or acidic side chain, and X K6 Ha, Yoshi a light chain variable region ("VL") comprising a nucleotide sequence (a region of interest where the nucleotide sequence is an amino acid having an aromatic side chain); VH CDR1, VH CDR2, VH CDR3, VH CDR4, VH CDR5, VH CDR6, VH CDR7, VH CDR8, VH CDR9, VH CDR10, VH CDR11, VH CDR12, VH CDR13, VH CDR14, VH CDR15, VH CDR16, VH CDR17, VH CDR18, VH CDR19, VH C a heavy chain variable region ("VH") comprising a VH CDR2, and a VH CDR3 The antibody or antigen-binding fragment thereof, or a conjugate thereof, comprises:

[0011] In certain embodiments, provided herein are antibodies that immunospecifically bind to D4 of human KIT. An antibody (or a fragment thereof or a conjugate thereof) comprising: (i) VL CDs having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively VL comprising R1, VL CDR2, and VL CDR3; and (ii) Amino acid sequence: [ka] (where X H1 is an amino acid with an aliphatic side chain, and X H2 is an amino group having an aliphatic side chain. X H3 is an amino acid with a polar or basic side chain, and X H4 has an aliphatic side chain X is an amino acid having H5 is an amino acid with an aliphatic side chain, and X H6 has an acidic side chain X is an amino acid having H7 is an amino acid having an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain) The antibody (or a fragment thereof or a conjugate thereof) comprises:

[0012] In certain embodiments, X K1 is an amino acid F or S, and X K2 is the amino acid A or S Ri, X K3 is the amino acid T or S, and X K4 is the amino acid S or P, and X K5 is the amino acid D or is T and X K6 is the amino acid F or Y.

[0013] In some embodiments, X K1 is the amino acid S and X K2 is the amino acid A and X K3 teeth, The amino acid T is X K4 is the amino acid P and X K5 is the amino acid D and X K6 is an amino acid It's F.

[0014] In certain embodiments, X K1 is the amino acid F and X K2 is the amino acid A and X K3 teeth , the amino acid T and X K4 is the amino acid S and X K5 is the amino acid D and X K6 is Amino Acid F.

[0015] In certain embodiments, XK1 is an amino acid F or S, and X K2 is the amino acid A and X K3 is the amino acid T and X K4 is the amino acid S or P, and X K5 is the amino acid D and X K6 is the amino acid F.

[0016] In certain embodiments, X K1 is the amino acid S and X K2 is the amino acid A and X K3 teeth , the amino acid T and X K4 is the amino acid P and X K5 is the amino acid D and X K6 is Amino Acid F.

[0017] In certain embodiments, X K1 is the amino acid S and X K2 is the amino acid S and X K3 teeth , amino acid S and X K4 is the amino acid P and X K5 is the amino acid T and X K6 is Amino Acid Y.

[0018] In one embodiment, X H1 is an amino acid L or V, and X H2 is an amino acid L or V, X H3 is the amino acid K or R, and X H4 is an amino acid V or A, and X H5 is amino acid L or I and X H6 is amino acid E or D, and X H7 is the amino acid Q or E, and X H8 is Amino Acid S or T.

[0019] In specific embodiments, XH1 is the amino acid V and X H2 is the amino acid L or V , X H3 is an amino acid R or Q, and X H4 is the amino acid A and X H5 is amino acid L or I Ri, X H6 is the amino acid D and X H7 is the amino acid Q or E, and X H8 is the amino acid T .

[0020] In specific embodiments, X H1 is the amino acid V and X H2 is the amino acid L and X H3 is the amino acid R and X H4 is the amino acid A and X H5 is the amino acid L and X H6 Ami Acid D and X H7 is the amino acid Q and X H8 is the amino acid T.

[0021] In some embodiments, X H1 is the amino acid V and X H2 is the amino acid V and X H3 teeth, Amino acid R and X H4 is the amino acid A and X H5 is amino acid I and X H6 is an amino acid D and X H7 is the amino acid E and X H8 is the amino acid T.

[0022] In some embodiments, X H1 is the amino acid L and X H2 is the amino acid L and X H3 teeth, Amino acid K and X H4 is the amino acid A and X H5 is the amino acid L and XH6 is an amino acid E and X H7 is the amino acid Q and X H8 is the amino acid S.

[0023] In some embodiments, X H1 is the amino acid V and X H2 is the amino acid L and X H3 teeth, Amino acid K and X H4 is the amino acid A and X H5 is the amino acid L and X H6 is an amino acid E and X H7 is the amino acid Q and X H8 is the amino acid T.

[0024] In some embodiments, X H1 is the amino acid V and X H2 is the amino acid V and X H3 teeth, Amino acid R and X H4 is the amino acid V and X H5 is amino acid I and X H6 is an amino acid D and X H7 is the amino acid E and X H8 is the amino acid T.

[0025] In certain embodiments, X K1 ~X K6 is an amino acid shown in Table 6A, and / or X H1 ~X H8 is an amino acid shown in Table 6B.

[0026] In certain embodiments, provided herein are antibodies that immunospecifically bind to D4 of human KIT. An antibody, or antigen-binding fragment thereof, or a conjugate thereof, comprising: i) at least 90% identical to SEQ ID NO: 7, at least 88% identical to SEQ ID NO: 8, and at least 90% identical to SEQ ID NO: 9 a VL comprising an amino acid sequence that is at least 87% identical, or at least 84% identical to SEQ ID NO: 10; and ii) at least 93% identical to SEQ ID NO:2, at least 92% identical to SEQ ID NO:3, and at least 92% identical to SEQ ID NO:4 at least 90% identical to SEQ ID NO: 5, at least 87% identical to SEQ ID NO: 5, or at least 86% identical to SEQ ID NO: 6 1. A VH comprising an amino acid sequence The antibody or antigen-binding fragment thereof, or a conjugate thereof, comprises:

[0027] In one embodiment, provided herein are antibodies that immunospecifically bind to the D4 region of human KIT. An antibody, or antigen-binding fragment thereof, or a conjugate thereof, which binds to: i) Amino acid sequence: [ka] (where X K1 is an amino acid having an aromatic or aliphatic hydroxyl side chain, and X K2 teeth, an amino acid having an aliphatic side chain or an aliphatic hydroxyl side chain, X K3 is an aliphatic hydrochloride an amino acid with a xyl side chain, X K4 is an amino acid with an aliphatic hydroxyl side chain or P and X K5 is an amino acid with a charged or acidic side chain, and X K6 Ha, Yoshi a light chain variable region ("VL") comprising a nucleotide sequence (a region of interest where the nucleotide sequence is an amino acid having an aromatic side chain); ii) Amino acid sequence: [ka] (where X H1 is an amino acid with an aliphatic side chain, and X H2 is an amino group having an aliphatic side chain. X H3is an amino acid with a polar or basic side chain, and X H4 has an aliphatic side chain X is an amino acid having H5 is an amino acid with an aliphatic side chain, and X H6 has an acidic side chain X is an amino acid having H7 is an amino acid having an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain) The antibody or antigen-binding fragment thereof, or a conjugate thereof, comprises:

[0028] In certain embodiments, X K1 ~X K6 is an amino acid shown in Table 6A, and / or X H1 ~X H8 is an amino acid shown in Table 6B.

[0029] In certain embodiments, the antibodies described herein are When the EC 50 It specifically binds to CHO cells recombinantly expressing wild-type KIT. In certain embodiments, the antibodies described herein are capable of expressing IgG as measured by flow cytometry. When measured, the EC 50 The recombinant D4 / D5 region of human KIT In certain embodiments, the antibodies described herein specifically bind to When measured, the IC was approximately 600 pM or less. 50 and inhibits tyrosine phosphorylation of KIT.

[0030] In a specific embodiment, the antibodies described herein are produced by at least one be expressed at a titer of at least 0.0 μg / mL, or at least 1.1 μg / mL, or at least 1.2 μg / mL can be done.

[0031] In certain embodiments, the antibodies described herein comprise a human light chain constant region and a human heavy chain constant region. In one embodiment, the human light chain constant region of the antibody described herein comprises and a human kappa light chain constant region. In certain embodiments, the human The gamma heavy chain constant region is a human gamma heavy chain constant region.

[0032] In certain embodiments, the antibodies described herein are human IgG1 or IgG4 antibodies. In embodiments, the antibodies described herein are antigen-binding fragments or Fab fragments. In certain embodiments, the antibodies described herein are antigen-binding fragments or Fab fragments. In some embodiments, the antibodies described herein are bispecific antibodies. In this case, the antibodies described herein are internalized by the cell.

[0033] In certain aspects, provided herein are compounds comprising a compound as described herein linked to an agent. In a specific embodiment, the antibody or KIT-binding fragment thereof is a conjugate comprising: The agent is a toxin. In certain embodiments, the toxin is abrin, ricin A, Pseudomonas aeruginosa, or the like. In one embodiment, the conjugate is an exotoxin, a cholera toxin, or a diphtheria toxin. The compound is internalized by the cell.

[0034] In some aspects, provided herein are conjugates as described herein and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0035] In another aspect, provided herein are antibodies and medicaments as described herein. A pharmaceutical composition comprising an acceptable carrier.

[0036] In certain embodiments, provided herein are VH chain regions of the antibodies described herein: A polynucleotide containing a nucleotide sequence encoding a VL chain region or both a VL chain region and a VH chain region It is leotide.

[0037] In specific embodiments, the polynucleotides provided herein (e.g., isolated The selected polynucleotide comprises SEQ ID NO: 22, 23, 24, 25, or 26 encoding the VH. In certain embodiments, the polynucleotides provided herein (e.g., isolated polynucleotides) The nucleotide sequence includes SEQ ID NO: 27, 28, 29, or 30, which encodes the VL. In the polynucleotides provided herein (e.g., isolated polynucleotides), The population of polynucleotides (e.g., a population of isolated polynucleotides) may comprise a VH. SEQ ID NO: 22, 23, 24, 25, or 26 encoding VL, and SEQ ID NO: 27, 28, 29 encoding VL; or 30 inclusive.

[0038] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 22 encoding the VH and SEQ ID NO: 27 encoding the VL.

[0039] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 22 encoding the VH and SEQ ID NO: 28 encoding the VL.

[0040] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 22 encoding the VH and SEQ ID NO: 29 encoding the VL.

[0041] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 22 encoding the VH and SEQ ID NO: 30 encoding the VL.

[0042] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 23 encoding the VH and SEQ ID NO: 27 encoding the VL.

[0043] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 23 encoding the VH and SEQ ID NO: 28 encoding the VL.

[0044] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 23 encoding the VH and SEQ ID NO: 29 encoding the VL.

[0045] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 23 encoding the VH and SEQ ID NO: 30 encoding the VL.

[0046] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 24 encoding the VH and SEQ ID NO: 27 encoding the VL.

[0047] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 24 encoding VH and SEQ ID NO: 28 encoding VL.

[0048] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 24 encoding the VH and SEQ ID NO: 29 encoding the VL.

[0049] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 24 encoding the VH and SEQ ID NO: 30 encoding the VL.

[0050] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 25 encoding VH and SEQ ID NO: 27 encoding VL.

[0051] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 25 encoding VH and SEQ ID NO: 28 encoding VL.

[0052] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 25 encoding VH and SEQ ID NO: 29 encoding VL.

[0053] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 25 encoding VH and SEQ ID NO: 30 encoding VL.

[0054] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 26 encoding VH and SEQ ID NO: 27 encoding VL.

[0055] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 26 encoding VH and SEQ ID NO: 28 encoding VL.

[0056] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 26 encoding VH and SEQ ID NO: 29 encoding VL.

[0057] In certain embodiments, the polynucleotides provided herein (e.g., isolated a population of polynucleotides (e.g., isolated polynucleotides) or a population of polynucleotides (e.g., isolated polynucleotides The population includes SEQ ID NO: 26 encoding the VH and SEQ ID NO: 30 encoding the VL.

[0058] In one aspect, provided herein is a method for expressing an anti-KIT antibody or fragment thereof. In one embodiment, the vector comprises a polynucleotide described herein for The vectors provided herein are mammalian expression vectors.

[0059] In some embodiments, provided herein are methods for expressing an anti-KIT antibody or fragment thereof. The vectors provided herein for The host cell contains the

[0060] In certain embodiments, provided herein are cells that produce the antibodies described herein. In one embodiment, the cells provided herein are one or more of the cells described herein. wherein the cell expresses an antibody that specifically binds to D4 of human KIT. In some embodiments, the cells are capable of expressing the vectors described herein. include.

[0061] In a specific embodiment, provided herein is an antibody (or In certain embodiments, the kit comprises a medicament for the treatment of a cancer, ... an antigen-binding fragment thereof, or a conjugate thereof. In the present invention, the kit comprises a conjugate described herein.

[0062] In some embodiments, provided herein are methods for treating or preventing a KIT-associated disorder (e.g., cancer). The method comprises administering to a subject in need thereof an antibody or antibody thereof as described herein. administering a therapeutically effective amount of a soluble binding fragment or a conjugate thereof to a subject. .

[0063] In one aspect, provided herein are methods for treating or managing a KIT-associated disorder. administering to a subject in need thereof a therapeutically effective amount of a conjugate described herein. The method includes:

[0064] In certain embodiments, the KIT-associated disorder is cancer, an inflammatory disease, or a fibrotic disease. In particular embodiments, the cancer is leukemia, chronic myeloid leukemia, lung cancer, small cell lung cancer, or In one embodiment, the cancer is a gastrointestinal stromal tumor. In a further embodiment, the tyrosine kinase inhibitor is methylprednisolone mesylate. Imatinib or SU11248.

[0065] In certain embodiments, the methods provided herein further comprise administering a second agent. In a specific embodiment, the second agent is a chemotherapeutic agent, a tyrosine kinase inhibitor, or In certain embodiments, the compound is a nucleotide analog, a histone deacetylase inhibitor, an antibody, or a cytokine. In one embodiment, the tyrosine kinase inhibitor is imatinib mesylate or SU11248.

[0066] In a specific embodiment, provided herein are methods for diagnosing a subject with a KIT-associated disorder. The method comprises treating cells or a sample obtained from the subject with an antibody (or or an antigen-binding fragment thereof or a conjugate thereof), and For example, detecting the expression level of KIT antigen present in the cell or sample. Detection of binding of the antibodies described herein to the KIT gene is indicative of the expression level of KIT in the cell or sample. In certain embodiments, the antibody can be correlated with a detectable molecule. In some embodiments, the detectable molecule is an enzyme, a fluorescent The molecule may be a luminescent molecule or a radioactive molecule.

[0067] In certain embodiments, provided herein are methods for detecting KIT in cells expressing KIT. a method for inhibiting the activity of the cells by treating the cells with an antibody (or antigen-binding The method comprises contacting the antibody with an effective amount of a compound selected from the group consisting of a nucleotide sequence of the antibody, ... fragment thereof, or a conjugate thereof.

[0068] In certain embodiments, provided herein are methods for the detection of antigens in cells expressing KIT. A method for inducing or enhancing apoptosis comprises treating the cells with an antibody (or the antigen-binding fragment thereof, or a conjugate thereof, It is the law.

[0069] In certain aspects, provided herein are methods of inducing cell differentiation, comprising: Cells expressing KIT were treated with an antibody (or antigen-binding fragment thereof) or In certain embodiments, the method comprises contacting the compound with an effective amount of a conjugate of In this case, the cells are stem cells.

[0070] In one embodiment, provided herein are antibodies that immunospecifically bind to the D4 region of human KIT. A method for producing an antibody against a leukemia virus comprising culturing a cell or host cell described herein. In one embodiment, provided herein is a method for detecting a target gene in the D4 region of human KIT. A method for producing an antibody that immunospecifically binds to a cell or In certain embodiments, the method comprises expressing the gene using a host cell. The cell is an isolated cell. In certain embodiments, the method comprises the step of: The method further comprises purifying the antibody obtained from

[0071] In one aspect, provided herein are antibodies that immunospecifically bind to the D4 region of human KIT. an antibody or antigen-binding fragment thereof comprising: (i) a light chain variant comprising a VL CDR1, a VL CDR2, and a VL CDR3 selected from the group shown in Tables 10 to 12; variable domain ("VL"); and (ii) a heavy chain comprising a VH CDR1, a VH CDR2, and a VH CDR3 selected from the group shown in Tables 13 to 15; Variable region ("VH") Includes:

[0072] In some aspects, described herein is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (i) a VL comprising VL FR1, VL FR2, VL FR3, and VL FR4 selected from the group shown in Tables 20 to 23; L; and (ii) VH FR1, VH FR2, VH FR3, and VH FR4 selected from the group shown in Tables 16 to 19. VH Includes:

[0073] In certain aspects, the antibodies or antigen-binding fragments described herein have amino acid modifications. In some embodiments, the antibodies or antigen-binding fragments described herein comprise an Fc region comprising an IgG1 antigen. In one embodiment, the antibody comprises an Fc region that is an IgG4 isotype or an IgG4 isotype. In certain embodiments, the antibody or antigen-binding fragment of the present invention is a humanized antibody. The antibody or antigen-binding fragment thereof is a bispecific antibody.

[0074] In some embodiments, the compounds described herein are conjugated to another agent. It is an antibody or an antigen-binding fragment thereof.

[0075] In one aspect, provided herein is an antibody or antigen-binding A composition comprising the fragment.

[0076] In certain aspects, provided herein are antibodies or antigens thereof described herein. VH chain region of an antigen-binding fragment (e.g., an antibody or antigen-binding fragment thereof comprising a sequence shown in Tables 10 to 15) a polynucleotide comprising a nucleotide sequence encoding a VL chain region, or both a VL chain region and a VH chain region; Also provided are vectors containing the polynucleotides described herein. In one embodiment, the vector is a mammalian expression vector.

[0077] In some embodiments, provided herein are one or more polynucleotides described herein. In one aspect, provided herein is a host cell comprising a vector of the invention. The antibodies or antigen-binding fragments thereof described herein (e.g., antibodies comprising the sequences shown in Tables 10 to 15) The term "antigen-binding fragment" refers to a cell that produces a specific antigen (antibody or antigen-binding fragment thereof).

[0078] In certain aspects, provided herein are antibodies or antigens thereof described herein. Kits containing binding fragments (e.g., antibodies or antigen-binding fragments thereof containing sequences shown in Tables 10 to 15) It is.

[0079] In some aspects, provided herein are methods for treating or managing a KIT-associated disorder. administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein (e.g., For example, a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising a sequence shown in Tables 10 to 15 is administered. In one embodiment, the KIT-associated disorder is cancer, an inflammatory disease, or In certain embodiments, the cancer is leukemia, chronic myeloid leukemia, lung cancer, Small cell lung cancer or gastrointestinal stromal tumor.

[0080] In certain embodiments, the methods of treating or managing a KIT-associated disorder described herein comprise a second In certain embodiments, the second agent is a chemotherapy agent. therapeutic agents, tyrosine kinase inhibitors, histone deacetylase inhibitors, antibodies, cytokines, The compound is an HSP90 inhibitor, a PGP inhibitor, or a proteosome inhibitor.

[0081] In one aspect, provided herein is a method for diagnosing a subject with a KIT-associated disorder. a method for detecting a cell or sample obtained from the subject, the cell or sample being treated with an antibody or antigen binding site thereof as described herein. contacting the antibody or antigen-binding fragment thereof with an antibody or antigen-binding fragment thereof comprising a sequence shown in Tables 10 to 15, and detecting the expression level of KIT in the cell or the sample. In an embodiment, the antibody is conjugated to a detectable molecule.

[0082] In one embodiment, provided herein are methods for detecting KIT activity in cells expressing KIT. a method for inhibiting the activity of a cell, the method comprising treating the cell with an antibody or antigen-binding fragment thereof described herein (e.g., For example, an antibody or antigen-binding fragment thereof comprising a sequence shown in Tables 10 to 15 is contacted with an effective amount of The method includes:

[0083] A method for inducing or enhancing apoptosis in cells expressing KIT, comprising: The cells are then subjected to immunization with an antibody or antigen-binding fragment thereof described herein (e.g., a fragment having a sequence shown in Tables 10-15). The method comprises contacting the subject with an effective amount of an antibody or antigen-binding fragment thereof comprising:

[0084] A method for producing an antibody that immunospecifically binds to the D4 region of human KIT, comprising: and culturing the cells and / or using the cells to express the antibody. [Brief explanation of the drawings]

[0085] (4. Brief description of the drawings) [Figure 1]Figure 1 shows the amino acid sequence of full-length human KIT (SEQ ID NO: 1), GenBank™ Accession No. AAC50969. The first five extracellular Ig-like domains (i.e., D1, D2, D3, D4, and D5) are shown; "{" indicates the amino-terminal residue of each domain, and "}" indicates the carboxyl-terminal residue of each domain. The D1 domain is located at P34 to R112, the D2 domain is located at D113 to P206, the D3 domain is located at A207 to D309, the D4 domain is located at K310 to N410 (SEQ ID NO: 15), the hinge region between D4 and D5 is located at V409 to N410, and the D5 domain is located at T411 to K509. The D1 / D2 hinge region is located between D113 and L117; the D2 / D3 hinge region is located between P206 and A210; and the D3 / D4 hinge region is located between D309 and G311. The D4 / D5 region includes K310 to K509. The transmembrane domain includes residues F525 to Q545, and the kinase domain includes residues K589 to S933.

[0086] [Figure 2] Figure 2 shows the amino acid sequence of recombinant KIT D4 / D5. Human KIT amino acids V308 to H515 (SEQ ID NO: 73) are shown in bold. The polypeptide shown (SEQ ID NO: 14) contains (i) the first 33 amino acids at the amino terminus of human KIT (i.e., M1 to E33) (underlined, including the non-bolded signal peptide), (ii) the D4 / D5 region of human KIT (bold), and (iii) a 5xHis tag at the carboxyl terminus (italicized).

[0087] [Figure 3A] Figure 3A shows the amino acid sequence of the H1 VH domain (SEQ ID NO:2) and the DNA encoding the amino acid sequence (SEQ ID NO:22). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0088] [Figure 3B]Figure 3B shows the amino acid sequence of the H2 VH domain (SEQ ID NO:3) and the DNA encoding the amino acid sequence (SEQ ID NO:23). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0089] [Figure 3C] Figure 3C shows the amino acid sequence of the H3 VH domain (SEQ ID NO:4) and the DNA encoding the amino acid sequence (SEQ ID NO:24). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0090] [Figure 3D] Figure 3D shows the amino acid sequence of the H4 VH domain (SEQ ID NO:5) and the DNA encoding the amino acid sequence (SEQ ID NO:25). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0091] [Figure 3E] Figure 3E shows the amino acid sequence of the H5 VH domain (SEQ ID NO:6) and the DNA encoding the amino acid sequence (SEQ ID NO:26). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0092] [Figure 3F]Figure 3F shows the amino acid sequence of the L1 VL domain (SEQ ID NO:7) and the DNA encoding the amino acid sequence (SEQ ID NO:27). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0093] [Figure 3G] Figure 3G shows the amino acid sequence of the L2 VL domain (SEQ ID NO:8) and the DNA encoding the amino acid sequence (SEQ ID NO:28). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0094] [Figure 3H] Figure 3H shows the amino acid sequence of the L3 VL domain (SEQ ID NO:9) and the DNA encoding the amino acid sequence (SEQ ID NO:29). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0095] [Figure 3I] Figure 3I shows the amino acid sequence of the L4 VL domain (SEQ ID NO: 10) and the DNA encoding the amino acid sequence (SEQ ID NO: 30). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (CDR1, CDR2, and CDR3) are shown. Both Kabat and numerical numbering of amino acid residues are shown.

[0096] [Figure 4] Figure 4A shows the consensus sequence of the VH domain (SEQ ID NO: 11). XH1-H8 indicate amino acids that may be any amino acid.

[0097] Figure 4B shows the consensus sequence of the VL domain (SEQ ID NO: 12). K1-K6 is any amino acid The amino acid may be an acid.

[0098] [Figure 5] 5 shows the binding activity of antibodies Hum17, Hum8, Hum4, and Hum10, and a chimera of antibody 37M ("chimera"), to a recombinant polypeptide of the D4 / D5 region of human KIT, as measured by solid-phase ELISA. The EC50 value for each antibody is shown.

[0099] [Figure 6] 6 shows a graph of the results of a binding assay performed by flow cytometry using CHO cells recombinantly expressing wild-type human KIT to characterize the KIT binding activity of antibodies Hum17, Hum8, Hum4, and Hum10 in comparison with a chimeric version of antibody 37M ("Chimera"). EC50 values ​​for each antibody are shown.

[0100] [Figure 7] 7 shows a graph of the results of a KIT phosphorylation inhibition assay performed by ELISA using CHO cells recombinantly expressing wild-type KIT to characterize the phosphorylation-blocking activity of antibodies Hum17, Hum8, Hum4, and Hum10 compared to a chimeric version of antibody 37M ("Chimera"). IC50 values ​​for each antibody are shown. DETAILED DESCRIPTION OF THE INVENTION

[0101] (5. Detailed Description) Unless otherwise defined, all technical and scientific terms used herein are understood by those of ordinary skill in the art. has the same meaning as commonly understood by

[0102] As used herein, the terms "about" or "approximately" , means within ±10% of a given value or range.

[0103] Provided herein are KIT polypeptides (e.g., KIT polypeptides containing the human KIT D4 domain). Antibodies and antigen-binding fragments thereof that immunospecifically bind to a specific polypeptide, as well as their components Also provided are conjugates encoding such antibodies and antigen-binding fragments thereof. Also provided are isolated nucleic acids (polynucleotides) that bind to such antibodies. or vectors (e.g., expression vectors) and cells containing nucleic acids encoding antigen-binding fragments thereof (e.g., host cells). Also provided are such antibodies, cells, e.g., host cells. Also provided herein are methods for producing cells for treating a KIT-associated disorder or disease ( for example, cancer, inflammatory disease, or fibrosis), or any such KIT-associated disorder or disease. Methods and uses for treating or managing one or more of the effects of one or more of the a method comprising administering an antibody or antigen-binding fragment thereof, or a conjugate thereof. Also provided herein are methods and uses for treating KIT-associated disorders or diseases (e.g., cancer, inflammation). a method for diagnosing a fibrotic disease (an inflammatory disease or fibrotic disease) comprising treating a sample with one or more antibodies (antibodies) described herein ( or an antigen-binding fragment thereof) and measuring the amount of IgG in the sample compared to a reference sample (e.g., a control sample). Further provided herein are methods for determining the expression level of KIT in a mammalian cell line. The present invention provides a method and use for inhibiting KIT activity in KIT-expressing cells, the method comprising: contacting a cell with an effective amount of an antibody or antigen-binding fragment thereof. Also provided herein are methods for promoting cell differentiation or differentiation in KIT-expressing cells. A method for inducing or enhancing apoptosis comprising treating the cells with an antibody or antibodies described herein. or a method comprising contacting the subject with an effective amount of antibody(ies).

[0104] As used herein, the term "D4 / D5 region" or "D4 / D5 domain" refers to a KI The fourth Ig-like extracellular ("D4") domain of T, the fifth Ig-like extracellular ("D5") domain, and the D4 The hinge region between the D4 and D5 domains (the "D4-D5 hinge region") contains the At the carboxyl end, in the following order: D4, the D4-D5 hinge region, and D5. As used herein, a region within the KIT polypeptide that includes the nucleotide sequence of FIG. The amino acids V308 to H515 and the polypeptide shown in Figure 2 are considered to be an example of the D4 / D5 region or domain. is.

[0105] As used herein, "KIT" or "KIT receptor" or "KIT polypeptide" refers to The term "KIT" refers to any form of full-length KIT, including native KIT, KIT isoforms, and the like. forms, interspecies KIT homologs, or KIT variants, e.g., naturally occurring variants (e.g., A. allelic or splice variants, or mutants, e.g., somatic mutants), or These include artificially constructed variants (e.g., recombinant or chemically modified variants), KIT is a type III receptor tyrosine kinase encoded by the c-kit gene. (See, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullrich and Schlessinge r, Cell, 1990, 61:203-212; Clifford et al., J. Biol. Chem., 2003, 278:314 61-31464; Yarden et al., EMBO J., 1987, 6:3341-3351; Mol et al., J. Biol. Chem ., 2003, 278:31461-31464). GenBank™ Accession No. NM_00022 2 provides an exemplary human KIT nucleic acid sequence. GenBank™ Accession No. NP_001087 241, P10721, and AAC50969 provide exemplary human KIT amino acid sequences. ) Accession No. AAH75716 provides an exemplary mouse KIT amino acid sequence. Active KIT contains five extracellular immunoglobulin (Ig)-like domains (D1, D2, D3, D4, and D5), a single membrane-associated domain, and Separated by a transmembrane region, an inhibitory cytoplasmic juxtamembrane domain, and a kinase insert segment It contains a split cytoplasmic kinase domain (see, e.g., Yarden et al., Nature, 1986, 3 23:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212; Clifford et al. (See J. Biol. Chem., 2003, 278:31461-31464). An exemplary amino acid sequence is shown in Figure 1 from amino acid residues V308 to H515. In certain embodiments, KIT is a human KIT. It can exist as a dimer, multimer, native form, or denatured form.

[0106] The term "fragment" as used herein in the context of a peptide or polypeptide The term refers to a peptide or polypeptide that contains less than the full-length amino acid sequence. The amino acid sequence may be, for example, an amino-terminal truncation, a carboxy-terminal truncation, and / or a sequence The fragment may result from an internal deletion of one or more residues from the sequence. In some embodiments, the KIT A fragment or antibody fragment (e.g., an antibody fragment that immunospecifically binds to a KIT polypeptide) may be KIT polypeptides or antibodies (e.g., antibodies that immunospecifically bind to KIT polypeptides), respectively. At least 5 consecutive amino acid residues of the amino acid sequence of amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues a group of at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, At least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least At least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least Also included are polypeptides containing an amino acid sequence of 250 consecutive amino acid residues. In some embodiments, a KIT polypeptide or antibody (e.g., an antibody that immunospecifically binds to a KIT polypeptide) is used. A fragment of a polypeptide or antibody (a fragment of a polypeptide or antibody containing at least one, at least two, or It has at least three functions.

[0107] As used herein, the term "host cell" refers to a particular cell that contains an exogenous nucleic acid molecule. cells, e.g., cells that have been transfected or transformed with a nucleic acid molecule, and such The progeny or potential progeny of such a parent cell. The progeny of such a cell may arise in subsequent generations. due to mutations or environmental influences occurring during the replication of the nucleic acid molecule, or due to integration of the nucleic acid molecule into the host cell genome, May not be identical to the parent cell.

[0108] (5.1 Antibodies) As used herein, the terms "antibody" and "immunoglobulin" and "Ig" are terms of art that may be used interchangeably herein and refer to antibodies that immunospecifically bind to an antigen. It refers to a molecule that has an antigen-binding site.

[0109] As used herein, an "antigen" includes an epitope and is therefore capable of being bound by an antibody. In a specific embodiment, the present invention provides a moiety or molecule that is specifically bound by the The antigen to which the antibodies described herein bind is KIT (e.g., human KIT), or a fragment thereof, e.g., KIT ( For example, the extracellular domain of KIT or the D4 region of KIT (eg, human KIT).

[0110] As used herein, "epitope" is a term of art and refers to a molecule to which an antibody specifically binds. The term "epitope" refers to a localized region of an antigen that can bind to a target molecule. An epitope may be a sequence of consecutive amino acids of a polypeptide, or an epitope may be a sequence of consecutive amino acids of a polypeptide. It may be a collection of two or more discontinuous regions.

[0111] As used herein, "antigen-binding domain," "antigen-binding region," "antigen-binding" The term "antigen fragment," and similar terms, refers to a fragment that interacts with an antigen and causes an antibody molecule to bind to the antigen. The portion of an antibody molecule that contains the amino acid residues that confer its specificity (e.g., the complementarity-determining region (CDR) The antigen-binding region may be derived from any animal species, e.g., rodent (e.g., mouse, rat, or The CDRs of the antibody molecule can be derived from either human or human origins (e.g., hamster). In particular, CDRs can be determined according to the Kabat numbering system. (Kabat et al., 1991), Sequences of Proteins of Immunological Interest of Immunological Interest)(US Department of Health and Human Services, Washin (See, e.g., Gillington, DC, 5th ed.). In some embodiments, the CDRs of the antibody are (i) those described herein. In the Chothia numbering scheme (e.g., Chothia and Lesk texts) called "Chothia CDRs" K., 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; and U.S. Patent No. 7,709,226); or (ii) see, for example, Lefranc, M.-P. 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids cids Res., 27:209-212. .

[0112] As used herein, a "conformational epitope" or "non-linear epitope" or A "discontinuous epitope" is an epitope that consists of at least two amino acids that are not consecutive in a single protein chain. This refers to an epitope consisting of two amino acids. For example, a conformational epitope is one that has an intervening separated by a stretch of amino acids, but Two or more amino acids that are close enough together to be recognized as a single epitope by an antibody As a further example, it may be composed of The amino acids present in the protein structure are either bound together or present on separate protein chains. are brought into close proximity due to the conformational shape of the structure or complex and are blocked by the anti-KIT antibodies described herein. Generally, the antigen-binding domains of the antibodies described herein can be conformational epitopes that can be bound by the antigen-binding domains. The linear epitopes bound by KIT antibodies are located in the secondary, tertiary, or quaternary structure of the KIT receptor. It will be understood by those skilled in the art that this may or may not depend on the For example, in some embodiments, the anti-KIT antibodies described herein are derived from the native three-dimensional protein. It binds to aggregates of amino acids, whether or not they are folded into a protein structure. In embodiments, the anti-KIT antibodies described herein recognize and bind to an epitope Therefore, rather than recognizing the individual amino acid residues that make up the epitope, (bending, twisting, turning, or folding).

[0113] As used herein, the term "constant region" or "constant domain" refers to the Although not directly involved in binding to antigens, they act through various effectors, such as through interactions with Fc receptors. It refers to functional portions of an antibody, such as the carboxyl-terminal portions of the light and / or heavy chains. These terms refer to the more generally conserved amino acids compared to immunoglobulin variable domains. It refers to a portion of an immunoglobulin molecule having a sequence.

[0114] As used herein, the term "heavy chain" when used in reference to an antibody refers to IgG subclasses, including IgG1, IgG2, IgG3, and IgG4, are classified as IgA, IgD, and IgE, respectively. Any of the amino acid sequences of the constant domains that give rise to antibodies of the gE, IgG, and IgM classes. Different types, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and It is referred to as mu (μ). In a specific embodiment, the heavy chain is a human heavy chain.

[0115] As used herein, "immunospecifically bind" and "immunospecifically recognize" The terms "specifically bind" and "specifically recognize" are used in the context of antibodies. and similar terms, and such binding will be understood by those skilled in the art to refer to binding to an antigen (e.g. It refers to a molecule that binds to a specific antigen (epitope, immune complex, or antigen-specific antigen). Molecules can be assayed using, for example, immunoassays, Biacore™, KinExA 3000 instruments (Sapidyne Instruments s, Boise, ID), or other assays known in the art. In particular embodiments, the peptide or polypeptide may bind to other peptides or polypeptides with a lower affinity than the peptide or polypeptide. A molecule that immunospecifically binds to an antigen has a K a Less than K at least 2 log, 2.5 log, 3 log, 4 log, or greater a binds to the antigen with another In a specific embodiment, molecules that immunospecifically bind to an antigen cross-link other proteins. In another specific embodiment, molecules that immunospecifically bind to an antigen do not react with other Does not cross-react with non-KIT proteins.

[0116] As used herein, an "isolated" or "purified" antibody refers to a substance from which the antibody is obtained. Substantially free from cellular material or other contaminating proteins derived from cellular or tissue sources or is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0117] The term "Kabat numbering" and similar terms are art-recognized and refer to antibodies or It refers to the system of numbering amino acid residues in the heavy and light chain variable regions of their antigen-binding portions (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391, and Kabat et al. (1991), Immunological Relationships Sequences of Proteins of Immunological Interest, 5th Edition , US Department of Health and Human Services, NIH Publication No. 91-3242). Ka Using the bat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31-35 ("CDR1"). , located at amino acid positions 50-65 ("CDR2"), and amino acid positions 95-102 ("CDR3"). Using the AT numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), It is located at amino acid positions 50 to 56 (CDR2) and amino acid positions 89 to 97 (CDR3).

[0118] As used herein, the term "light chain" when used in reference to an antibody refers to Any of the different types, such as kappa (κ) or lambda (L) based on the amino acid sequence of the constant domain. The light chain amino acid sequence is known in the art. wherein the light chain is a human light chain.

[0119] As used herein, the term "monoclonal antibody" refers to a homogeneous or substantially A monoclonal antibody is an antibody obtained from a population of antibodies that are generally homogeneous. Each monoclonal antibody is usually The term "monoclonal" refers to any particular antibody that recognizes a single epitope on the antigen. The method of production of monoclonal antibodies is not limited to the method of production. Typically, a population of monoclonal antibodies is produced from a cell, a population of cells, or In a specific embodiment, the term "mono- A "clonal antibody" refers to a single hybridoma or other cell (e.g., a cell line that produces a recombinant antibody). and antibodies produced by a host cell that is capable of detecting the antibody, for example, by ELISA or other methods known in the art. Other antigen binding or competitive binding methods known in the art or in the examples provided herein As measured by the assay, the immune response to a KIT epitope (e.g., the epitope of D4 of human KIT) The monoclonal antibodies described herein specifically bind to the antibodies described, for example, in Kohler et al. It can be prepared by the hybridoma method described in Nature, 256:495 (1975). or isolated from a phage library, for example, using the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are also possible. Methods are well known in the art (e.g., Short Protocols in Molecular Biology). ols in Molecular Biology (2002), 5th ed., Ausubel et al., John Wiley and Sons, New York rk: Chapter 11).

[0120] As used herein, the term "polyclonal antibody" refers to a polyclonal antibody that is a polyclonal antibody to an antigen (singular) or Various different antibodies directed against the same epitope and against different epitopes within the antigen(s) Polyclonal antibodies refer to a population of antibodies comprising: For example, Short Protocols in Molecular Biology (2002) , 5th ed., Ausubel et al., eds., John Wiley and Sons, New York: see Chapter 11).

[0121] As used herein, the term "recombinant human antibody" refers to an antibody produced solely by recombinant means. Human antibodies isolated, prepared, expressed, or produced, e.g., in trans in host cells. antibodies expressed using recombinant expression vectors transfected therein, recombinant combinatorial antibodies, Antibodies isolated from a human antibody library, transgenic for human immunoglobulin genes Animals that are genic and / or transchromosomal (e.g., mice, rabbits, Antibodies isolated from human (eg, guinea pig, or bovine) (e.g., Taylor, LD et al. (1992) Nucl. Ac ids Res. 20:6287-6295), or, for example, synthetic, encoding human immunoglobulin sequences. Genetic modification of DNA sequences encoding human immunoglobulins, e.g., human immunoglobulins. Any method involving the production of immunoglobulin gene sequences by splicing into other such sequences. This includes antibodies prepared, expressed, produced, or isolated by any other means. Such recombinant human antibodies contain variable and constant regions derived from human germline immunoglobulin sequences. In some embodiments, the amino acid sequence of such a recombinant human antibody may be The sequences are so modified and therefore the sequences of the VH and / or VL regions of the recombinant antibody are The amino acid sequences are derived from and related to human germline VH and VL sequences, but are not specific to in vivo These are sequences that do not naturally occur in the human antibody germline repertoire. Recombinant human antibodies are made by assembling several human sequence fragments into the synthetic human sequence of a recombinant human antibody. This can be obtained by

[0122] As used herein, the term "variable region" or "variable domain" refers to the The sequences vary widely among different antigens, which affects the binding and specificity of a particular antibody to its particular antigen. The portion of the antibody used, usually a portion of the light or heavy chain, typically an amino acid in the mature heavy chain. This refers to approximately 110-120 amino acids at the terminal end and approximately 90-100 amino acids in the mature light chain. The most abundant amino acids are concentrated in regions called complementarity-determining regions (CDRs), while the most abundant amino acids are concentrated in the variable domains. Highly conserved regions are called framework regions (FR). Without wishing to be bound by theory, the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In a specific embodiment, the amino acid sequences of the antibodies described herein are The numbering of the acid positions follows that of Kabat et al. (1991), the sequence of the protein of immunological interest (Sequence of Proteins of Immunological Interest), 5th edition, US Department of Health and H. Human Services, NIH Publication No. 91-3242 (Kabat et al., 2002). In some embodiments, the CDRs of an antibody are (i) those described herein as The Chothia numbering scheme, referred to as the "Chothia CDR" (e.g., Chothia and Lesk, 1999) 87, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:92 7-948; and U.S. Pat. No. 7,709,226; or (ii) see, for example, Lefranc, M.-P., 199 9, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Re s., 27:209-212. In some embodiments, the variable region is a human variable region. The regions comprise rodent or mouse CDRs and human framework regions (FR). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In the present invention, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) frames. As a non-limiting example, the variable regions described herein may be composed of human sequences It is obtained by assembling two or more fragments of the above into a synthetic human sequence.

[0123] In specific embodiments, provided herein are D4 of human KIT, and KIT, e.g., Antibodies (including antigen-binding fragments thereof) that immunospecifically bind to the D4 / D5 region of human KIT, e.g., The amino acid residues V308 to H515 (SEQ ID NO: 73) in Figures 1 and 2 are an exemplary sequence of human KIT. The amino acids K310 to N410 (SEQ ID NO: 15) shown in Figures 1 and 2 represent the D4 / D5 region of human KIT. In another specific embodiment, the antibody (or its antibody) described herein is The original binding fragment binds to KIT, e.g., the D4 domain of human KIT, with lower affinity than the D4 domain of human KIT. For example, immunospecifically binds to the D5 domain of human KIT. The antibodies (or antigen-binding fragments thereof) described herein are directed against the D5 domain of KIT, e.g., human KIT. immunospecifically binds to the D4 domain of KIT, e.g., human KIT, with higher affinity than For example, higher affinity can be measured by methods known in the art, such as ELISA or Biacore assays. When measured by the NMR spectroscopy, it is at least 10 times, 20 times, 50 times, 100 times, 500 times, or 1000 times.

[0124] In specific embodiments, the antibodies (or antigen-binding fragments thereof) described herein bind to KIT, For example, immunospecific binding to the D4 or D4 / D5 region of human KIT, which consists essentially of the D5 domain alone, has a higher affinity for a KIT antigen consisting essentially of only the D4 domain than for a KIT antigen consisting essentially of only the D4 domain In certain embodiments, the antibodies (or antigen-binding fragments thereof) described herein bind to KIT, e.g. For example, a nucleotide sequence that immunospecifically binds to the D4 or D4 / D5 region of human KIT and consists essentially of only the D5 domain. a D4 domain alone that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher than the KIT antigen In certain embodiments, the antibody has an affinity for the KIT antigen. The antibody (or antigen-binding fragment thereof) is immunospecific for the D4 or D4 / D5 region of KIT, e.g., human KIT. and a KIT antigen consisting essentially of only the D4 domain or D5 domain, which binds to the KIT antigen and is higher than the KIT antigen consisting essentially of only the D5 domain. Binding affinity to the KIT antigen consisting essentially of only the D4 / D5 region (e.g., approximately 2- to 3-fold higher affinity) (sexuality).

[0125] In certain embodiments, the antibody (or antigen-binding fragment thereof) described herein is selected from the group consisting of SEQ ID NO: A nucleotide sequence encoding a nucleotide sequence encoding a KIT antigen comprising or consisting essentially of a nucleotide sequence encoding a nucleotide sequence encoding a KIT antigen comprising a nucleotide sequence encoding ... KIT antigen. In specific embodiments, the antibodies (or antigen-binding fragments thereof) described herein are In certain embodiments, the antibody immunospecifically binds to the D4 domain of KIT, e.g., human KIT. The antibodies described herein are directed against a KIT antigen comprising or consisting essentially of D4 of human KIT. In certain embodiments, the antibodies described herein (or their antibodies) immunospecifically bind to The original binding fragment comprises the amino acid sequence of SEQ ID NO: 14 or 73 or is derived from the amino acid sequence. It immunospecifically binds to the KIT antigen, which is a qualitative antigen.

[0126] In certain embodiments, provided herein are KIT polypeptides (e.g., KIT, e.g., For example, the D4 region of human KIT (e.g., SEQ ID NO: 15 [human D4 sequence]) is immunospecifically bound to the D4 region of human KIT. The antibody or antigen-binding fragment thereof comprises an amino acid sequence as described herein.

[0127] In specific embodiments, described herein are: (i) [ka] and a VH CDR of a VH domain comprising the amino acid sequence (ii) [ka] an antibody (e.g., a human antibody) comprising an antigen-binding fragment thereof, the antibody comprising a VL CDR of a VL domain comprising the amino acid sequence of For example, a human or humanized antibody.

[0128] In a specific embodiment, the antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies (e.g., human or humanized antibodies) described herein that bind to In a specific embodiment, the KIT polypeptide comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence of the nucleotides 16 to 18 and a nucleotide sequence of the nucleotides 19 to 21. The antibodies (e.g., antibodies described herein) that immunospecifically bind to a polypeptide (e.g., the D4 region of human KIT). For example, a human or humanized antibody may have the VH CDRs and VL CDRs set forth in Table 2 (e.g., Set 1 or Set 2). In one embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) is immunized with the KIT polypeptide. Antibodies (e.g., human or humanized antibodies) described herein that specifically bind to the V It comprises the H CDRs and the VL CDRs (AbM CDRs or contact CDRs).

[0129] Table 1: CDR amino acid sequences [Table 1]

[0130] Table 2: CDR amino acid sequences [Table 2] Table 3: CDR amino acid sequences [Table 3]

[0131] Table 4: VL and VH domains of antibodies Hum1-20 [Table 4]

[0132] In some embodiments, provided herein are VH domains (e.g., VH domains of the sequences H1, H2, H3, H4, and H5, including numbers 2-6), and VL domains (e.g., sequences In some embodiments, the nucleotides provided herein are L1, L2, L3, and L4, including numbers 7-10. Examples of antibodies include antibodies comprising VH and VL domains as shown in Table 4 (i.e., antibodies In certain embodiments, these antibodies are selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 1 to 3, including VH CDRs 1 to 3 and VL CDRs 1 to 3, including 1 to 18 and 19 to 21.

[0133] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are The amino acid sequences described above, for example, any of SEQ ID NOs: 7 to 10 (see, for example, Figures 3F to 3I) or SEQ ID NO: 12 The variable light (VL) chain region comprises one of:

[0134] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are The amino acid sequence of any of the above, for example, SEQ ID NOs: 2 to 6 (see, for example, Figures 3A to 3E) or SEQ ID NO: 11 The variable heavy (VH) chain region comprises one of:

[0135] For example, described herein are antibodies that are immunized with a KIT polypeptide (e.g., the D4 region of human KIT). (i) VH domains H1 (SEQ ID NO: 2), H2 (SEQ ID NO: 3), H3 (SEQ ID NO: 4), H4 ( SEQ ID NO: 5), or H5 (SEQ ID NO: 6), and / or (ii) VL domains L1 (SEQ ID NO: 7), L2 (SEQ ID NO: 8), SEQ ID NO: 8), L3 (SEQ ID NO: 9), or L4 (SEQ ID NO: 10). The antibodies or antigen-binding fragments thereof described herein may be directed to a KIT polypeptide (e.g., the D of human KIT). 4 region), and binds immunospecifically to the VH domain and / or VL domain of any one of antibodies Hum1 to Hum20. In certain instances, the antibodies or domains described herein may comprise The antigen-binding fragment comprises the VH domain and / or VH domains of any one of antibodies Hum4, Hum8, Hum10, or Hum17. or a VL domain.

[0136] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The binding antibodies include H1 (SEQ ID NO: 2) and L1 (SEQ ID NO: 7). Antibodies that immunospecifically bind to a T polypeptide (e.g., the D4 region of human KIT) include H1 (SEQ ID NO:2 ) and L2 (SEQ ID NO: 8). In a specific embodiment, the KIT polypeptide (e.g., human Antibodies that immunospecifically bind to the D4 region of KIT include H1 (SEQ ID NO: 2) and L3 (SEQ ID NO: 9). In a specific embodiment, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). Binding antibodies include H1 (SEQ ID NO: 2) and L4 (SEQ ID NO: 10). Antibodies that immunospecifically bind to a KIT polypeptide (e.g., the D4 region of human KIT) include those that bind to H2 (sequence In a specific embodiment, the KIT polypeptide (e.g., Antibodies that immunospecifically bind to the D4 region of human KIT include H2 (SEQ ID NO: 3) and L2 (SEQ ID NO: 8). In a specific embodiment, a KIT polypeptide (e.g., the D4 region of human KIT) is immunospecific. Antibodies that bind heterologously include H2 (SEQ ID NO: 3) and L3 (SEQ ID NO: 9). In this case, antibodies that immunospecifically bind to a KIT polypeptide (e.g., the D4 region of human KIT) bind to H2( In a specific embodiment, the KIT polypeptide (e.g., L4) is a nucleotide sequence of the ... For example, antibodies that immunospecifically bind to the D4 region of human KIT include H3 (SEQ ID NO: 4) and L1 (SEQ ID NO: 5). In a specific embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) is immunized. Antibodies that immunospecifically bind include H3 (SEQ ID NO: 4) and L2 (SEQ ID NO: 8). wherein an antibody that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) is In a specific embodiment, the KIT polypeptide ( For example, antibodies that immunospecifically bind to the D4 region of human KIT include H3 (SEQ ID NO: 4) and L4 (SEQ ID NO: 5). No. 10). In a specific embodiment, a KIT polypeptide (e.g., the D4 region of human KIT) Antibodies that immunospecifically bind to include H4 (SEQ ID NO: 5) and L1 (SEQ ID NO: 7). In embodiments, an antibody that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT). In a specific embodiment, the KIT polypeptide comprises H4 (SEQ ID NO: 5) and L2 (SEQ ID NO: 8). Antibodies that immunospecifically bind to the D4 region of human KIT include H4 (SEQ ID NO: 5) and L3 ( In a specific embodiment, the KIT polypeptide (e.g., D4 of human KIT) Antibodies that immunospecifically bind to the H4 (SEQ ID NO: 5) and L4 (SEQ ID NO: 10) regions include H4 (SEQ ID NO: 5) and L4 (SEQ ID NO: 10). In certain embodiments, antibodies that immunospecifically bind to a KIT polypeptide (e.g., the D4 region of human KIT) are In a specific embodiment, the antibody comprises H5 (SEQ ID NO: 6) and L1 (SEQ ID NO: 7). Antibodies that immunospecifically bind to a polypeptide (e.g., the D4 region of human KIT) include H5 (SEQ ID NO: 6) and and L2 (SEQ ID NO: 8). In a specific embodiment, the KIT polypeptide (e.g., human KIT Antibodies that immunospecifically bind to the D4 region of the HIV-1 virus include H5 (SEQ ID NO: 6) and L3 (SEQ ID NO: 9). In a specific embodiment, the antibody binds immunospecifically to a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies involved include H5 (SEQ ID NO: 6) and L4 (SEQ ID NO: 10).

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof is non-immunogenic in humans. In certain embodiments, the non-immunogenic amino acid sequence is a binder to human MHC class II. epitopes identified as being specific for human MHC class II, e.g., non-human germline In certain embodiments, the amino acid sequence is epitopes identified as binders to human MHC class II, e.g., human Substantially devoid of epitopes that are non-human germline binders to human MHC class II. For example, to locate both B-cell and T-cell epitopes and to identify immunoglobulins. In silico tools have been developed to assess epidemiological potential. Such tools provide alternatives to in vitro or in vivo immunogenicity assays. For example, computational epitope prediction methods and experimentally derived epitopes are provided. Manually processed databases containing the data have been developed (Bryson et al., Biodr. ugs, 2010, 24(1): 1-8). Non-limiting examples of epitope databases include The Immune Epitope Database (IEDB) and the dedicated T Cell Epitope Database™ (TCED™) Such epitope databases can be used alone or in combination with other databases described in the art. In vitro assays such as MHC class II binding assays and T cell activation assays are also described. Alternatively, such in vitro assays can be used in conjunction with proliferation assays. The antibody assay can be used independently of such epitope databases. for determining the immunogenicity of an agent, such as a human antibody, or for depleting or eliminating the immunogenicity of an agent, such as an antibody. Methods for reducing the risk of thrombin generation have been described in the art, e.g., Altschul et al., Nucl eic Acids Res., 1997, 25:3389-3402; Baker et al., Curr. Opin. Drug Discov. Deve l., 2007, 10:219; Hill et al., Arthritis Res. Ther., 2003, 1:R40-R48; Jones et al. J. Thromb. Haemost., 2005, 3:991-1000; Holgate et al., IDrugs, 2009, 12:2 33-237; Jones et al., Methods Mol. Biol., 2009, 525:405-423; and Baker et al., See Curr. Drug Saf., 2010, 5:308-313. In certain embodiments, human KIT The antibodies described herein that immunospecifically bind to the D4 region of T Cell Epitope Database (T Cell Epitope Database) are listed in Table 1. The VH domain and VL domain are not immunogenic as determined by TCED™. In certain embodiments, the antibodies described herein immunospecifically target the D4 region of human KIT. binds to and is not immunogenic as determined by in vitro assays described in the art The VH domain and VL domain are not included in the nucleotide sequence of the present invention, and are described, for example, in Wang et al., 2008, PLoS Computation nal Biology, 2008, 4(4):e1000048; and Arnold et al., 2002, J. Immunol., 169:739- See 749.

[0138] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region) or its antigen-binding A fragment comprising a VH domain having at least 93% sequence identity with H1 (SEQ ID NO: 2). In certain embodiments, the antibody is an antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. In such a manner, antibodies that immunospecifically bind to a KIT polypeptide (e.g., the D4 region of human KIT) , H1 (SEQ ID NO: 2), or at least 94%, or at least 95%, or at least 96%, or VH domains with at least 97%, or at least 98%, or at least 99% sequence identity In certain embodiments, the VH domain comprises a VH domain that binds, for example, to MHC class II. epitopes, e.g., determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or their antigen binding sites are non-immunogenic. The combined fragments comprise VH CDR1, VH CDR2, and VH CDR3, each containing the amino acid sequences of SEQ ID NOs: 16 to 18. It contains a VH domain including DR3.

[0139] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and H2 (SEQ ID NO: 3). An antibody or antigen-binding fragment thereof comprising a VH domain having at least 92% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to H2 (SEQ ID NO: 3) has at least 93% affinity with H2 (SEQ ID NO: 3), or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least In certain embodiments, the VH domains have at least 98%, or at least 99%, sequence identity. In this case, the VH domain may, for example, bind to an epitope that binds to MHC class II, e.g., MHC class II. It is non-immunogenic as determined by the absence of non-human germline binders to serotype II. In certain embodiments, such an antibody or antigen-binding fragment thereof has the sequence of SEQ ID NO: 16, respectively. It contains a VH domain comprising VH CDR1, VH CDR2, and VH CDR3, which comprise a sequence of ∼18 amino acids.

[0140] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and An antibody or antigen-binding fragment thereof comprising a VH domain having at least 90% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to H3 (SEQ ID NO: 4) has at least 92% affinity to H3 (SEQ ID NO: 4), At least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% %, or at least 98%, or at least 99% sequence identity. In certain embodiments, the VH domain comprises an epitope that binds to, for example, MHC class II. , e.g., as determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or antigen-binding fragments thereof are VH CDR1, VH CDR2, and VH CDR3 each containing the amino acid sequences of SEQ ID NOs: 16 to 18. Includes the domain.

[0141] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and An antibody or antigen-binding fragment thereof comprising a VH domain having at least 87% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to H4 (SEQ ID NO: 5) has at least 92% affinity to H4 (SEQ ID NO: 5), At least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% %, or at least 98%, or at least 99% sequence identity. In certain embodiments, the VH domain comprises an epitope that binds to, for example, MHC class II. , e.g., as determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or antigen-binding fragments thereof are VH CDR1, VH CDR2, and VH CDR3 each containing the amino acid sequences of SEQ ID NOs: 16 to 18. Includes the domain.

[0142] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and H5 (SEQ ID NO: 6). An antibody or antigen-binding fragment thereof comprising a VH domain having at least 86% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to H5 (SEQ ID NO: 6) has at least 92% affinity to H5 (SEQ ID NO: 6), At least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% %, or at least 98%, or at least 99% sequence identity. In certain embodiments, the VH domain comprises an epitope that binds to, for example, MHC class II. , e.g., as determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or antigen-binding fragments thereof are VH CDR1, VH CDR2, and VH CDR3 each containing the amino acid sequences of SEQ ID NOs: 16 to 18. Includes the domain.

[0143] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and L1 (SEQ ID NO: 7). An antibody or antigen-binding fragment thereof comprising a VL domain having at least 90% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to L1 (SEQ ID NO: 7) has at least 92% affinity with L1 (SEQ ID NO: 7), At least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% %, or at least 98%, or at least 99% sequence identity. In certain embodiments, the VL domain comprises an epitope that binds, for example, MHC class II. , e.g., as determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or antigen-binding fragments thereof are VL CDR1, VL CDR2, and VL CDR3 each containing the amino acid sequences of SEQ ID NOs: 19 to 21. Includes the domain.

[0144] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and L2 (SEQ ID NO: 8). An antibody or antigen-binding fragment thereof comprising a VL domain having at least 88% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are Antibodies that bind to L2 (SEQ ID NO: 8) have at least 92%, at least 93%, at least 94% %, or at least 95%, or at least 96%, or at least 97%, or at least 98% %, or at least 99% sequence identity. The VL domain may, for example, encode an epitope that binds to MHC class II, e.g., MHC class II It is non-immunogenic as determined by the absence of non-human germline binders to the In embodiments, such antibodies or antigen-binding fragments thereof are selected from the group consisting of SEQ ID NOs: 19-21, respectively. and a VL domain comprising a VL CDR1, a VL CDR2, and a VL CDR3 comprising the amino acid sequences:

[0145] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and L3 (SEQ ID NO: 9). An antibody or antigen-binding fragment thereof comprising a VL domain having at least 87% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to L3 (SEQ ID NO: 9) has at least 92% affinity with L3 (SEQ ID NO: 9), At least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% %, or at least 98%, or at least 99% sequence identity. In certain embodiments, the VL domain comprises an epitope that binds, for example, MHC class II. , e.g., as determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or antigen-binding fragments thereof are VL CDR1, VL CDR2, and VL CDR3 each containing the amino acid sequences of SEQ ID NOs: 19 to 21. Includes the domain.

[0146] In some embodiments, provided herein are KIT polypeptides (e.g., D of human KIT). 4 region), and L4 (SEQ ID NO: 10). An antibody or antigen-binding fragment thereof comprising a VL domain having at least 84% sequence identity. In certain embodiments, antibodies immunospecific for a KIT polypeptide (e.g., the D4 region of human KIT) are The antibody or antigen-binding fragment thereof that binds to L4 (SEQ ID NO: 10) has at least 92% affinity with L4 (SEQ ID NO: 10), At least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97% %, or at least 98%, or at least 99% sequence identity. In certain embodiments, the VL domain comprises an epitope that binds, for example, MHC class II. , e.g., as determined by the absence of non-human germline binders to MHC class II. In certain embodiments, such antibodies or antigen-binding fragments thereof are VL CDR1, VL CDR2, and VL CDR3 each containing the amino acid sequences of SEQ ID NOs: 19 to 21. Includes the domain.

[0147] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 93% or at least 95% of that of H1 (SEQ ID NO: 2); and (ii) a VH domain comprising an amino acid sequence having at least 90% identity with L1 (SEQ ID NO: 7). an antibody comprising a VL domain comprising an amino acid sequence having 92% or at least 92% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0148] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 92% or at least 94% of H2 (SEQ ID NO: 3); and (ii) a VH domain comprising an amino acid sequence having at least 90% identity with L1 (SEQ ID NO: 7). an antibody comprising a VL domain comprising an amino acid sequence having 92% or at least 92% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0149] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence which shares at least 90% or at least 92% of the sequence of H3 (SEQ ID NO: 4); and (ii) a VH domain comprising an amino acid sequence having at least 90% identity with L1 (SEQ ID NO: 7). an antibody comprising a VL domain comprising an amino acid sequence having 92% or at least 92% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0150] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 87% or at least 90% of that of H4 (SEQ ID NO: 5); and (ii) a VH domain comprising an amino acid sequence having at least 90% identity with L1 (SEQ ID NO: 7). an antibody comprising a VL domain comprising an amino acid sequence having 92% or at least 92% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0151] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence that is at least 86% or at least 88% identical to that of H5 (SEQ ID NO: 6); and (ii) a VH domain comprising an amino acid sequence having at least 90% identity with L1 (SEQ ID NO: 7). an antibody comprising a VL domain comprising an amino acid sequence having 92% or at least 92% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0152] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 93% or at least 95% of that of H1 (SEQ ID NO: 2); and (ii) a VH domain comprising an amino acid sequence having at least 88 identical sequences with L2 (SEQ ID NO: 8). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0153] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 92% or at least 94% of H2 (SEQ ID NO: 3); and (ii) a VH domain comprising an amino acid sequence having at least 88 identical sequences with L2 (SEQ ID NO: 8). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0154] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence which shares at least 90% or at least 92% of the sequence of H3 (SEQ ID NO: 4); and (ii) a VH domain comprising an amino acid sequence having at least 88 identical sequences with L2 (SEQ ID NO: 8). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0155] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 87% or at least 90% of that of H4 (SEQ ID NO: 5); and (ii) a VH domain comprising an amino acid sequence having at least 88 identical sequences with L2 (SEQ ID NO: 8). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0156] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence that is at least 86% or at least 88% identical to that of H5 (SEQ ID NO: 6); and (ii) a VH domain comprising an amino acid sequence having at least 88 identical sequences with L2 (SEQ ID NO: 8). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0157] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 93% or at least 95% of that of H1 (SEQ ID NO: 2); and (ii) a VH domain comprising an amino acid sequence having at least 87 identical sequences with L3 (SEQ ID NO: 9). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0158] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 92% or at least 94% of H2 (SEQ ID NO: 3); and (ii) a VH domain comprising an amino acid sequence having at least 87 identical sequences with L3 (SEQ ID NO: 9). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0159] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence which shares at least 90% or at least 92% of the sequence of H3 (SEQ ID NO: 4); and (ii) a VH domain comprising an amino acid sequence having at least 87 identical sequences with L3 (SEQ ID NO: 9). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0160] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 87% or at least 90% of that of H4 (SEQ ID NO: 5); and (ii) a VH domain comprising an amino acid sequence having at least 87 identical sequences with L3 (SEQ ID NO: 9). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0161] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence that is at least 86% or at least 88% identical to that of H5 (SEQ ID NO: 6); and (ii) a VH domain comprising an amino acid sequence having at least 87 identical sequences with L3 (SEQ ID NO: 9). an antibody comprising a VL domain comprising an amino acid sequence having 90% or at least 90% sequence identity with (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. The VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC class II. Non-immunogenic as determined by the absence of non-human germline binders to Ras II In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence set forth in SEQ ID NO: VL CDRs 1 to 3 containing amino acid sequences of SEQ ID NOs: 19 to 21 and the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. and VH CDRs 1 to 3 comprising:

[0162] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 93% or at least 95% of that of H1 (SEQ ID NO: 2); and (ii) a VH domain comprising an amino acid sequence having at least 8 identical sequences with L4 (SEQ ID NO: 10). an antibody comprising a VL domain having an amino acid sequence having 4% or at least 86% sequence identity thereto; In certain embodiments, the antibody is a human antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. In this case, the VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC Non-immunogenic as determined by the absence of non-human germline binders to class II. In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence number VL CDRs 1 to 3 containing the amino acid sequences of SEQ ID NOs: 19 to 21 and VL CDRs 2 to 3 containing the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. Contains VH CDRs 1-3, including the sequence.

[0163] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 92% or at least 94% of H2 (SEQ ID NO: 3); and (ii) a VH domain comprising an amino acid sequence having at least 8 identical sequences with L4 (SEQ ID NO: 10). an antibody comprising a VL domain having an amino acid sequence having 4% or at least 86% sequence identity thereto; In certain embodiments, the antibody is a human antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. In this case, the VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC Non-immunogenic as determined by the absence of non-human germline binders to class II. In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence number VL CDRs 1 to 3 containing the amino acid sequences of SEQ ID NOs: 19 to 21 and VL CDRs 2 to 3 containing the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. Contains VH CDRs 1-3, including the sequence.

[0164] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence which shares at least 90% or at least 92% of the sequence of H3 (SEQ ID NO: 4); and (ii) a VH domain comprising an amino acid sequence having at least 8 identical sequences with L4 (SEQ ID NO: 10). an antibody comprising a VL domain having an amino acid sequence having 4% or at least 86% sequence identity thereto; In certain embodiments, the antibody is a human antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. In this case, the VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC Non-immunogenic as determined by the absence of non-human germline binders to class II. In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence number VL CDRs 1 to 3 containing the amino acid sequences of SEQ ID NOs: 19 to 21 and VL CDRs 2 to 3 containing the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. Contains VH CDRs 1-3, including the sequence.

[0165] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence identical to at least 87% or at least 90% of that of H4 (SEQ ID NO: 5); and (ii) a VH domain comprising an amino acid sequence having at least 8 identical sequences with L4 (SEQ ID NO: 10). an antibody comprising a VL domain having an amino acid sequence having 4% or at least 86% sequence identity thereto; In certain embodiments, the antibody is a human antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. In this case, the VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC Non-immunogenic as determined by the absence of non-human germline binders to class II. In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence number VL CDRs 1 to 3 containing the amino acid sequences of SEQ ID NOs: 19 to 21 and VL CDRs 2 to 3 containing the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. Contains VH CDRs 1-3, including the sequence.

[0166] In specific embodiments, provided herein are KIT polypeptides (e.g., human an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of human KIT or An antigen-binding fragment, comprising: (i) a sequence that is at least 86% or at least 88% identical to that of H5 (SEQ ID NO: 6); and (ii) a VH domain comprising an amino acid sequence having at least 8 identical sequences with L4 (SEQ ID NO: 10). an antibody comprising a VL domain having an amino acid sequence having 4% or at least 86% sequence identity thereto; In certain embodiments, the antibody is a human antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof. In this case, the VL and VH domains may, for example, bind to epitopes that bind to MHC class II, e.g., MHC Non-immunogenic as determined by the absence of non-human germline binders to class II. In certain embodiments, such antibodies or antigen-binding fragments thereof each have the sequence number VL CDRs 1 to 3 containing the amino acid sequences of SEQ ID NOs: 19 to 21 and VL CDRs 2 to 3 containing the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. VH CDRs 1-3 containing the sequence.

[0167] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are Align for optimal comparison purposes (e.g., optimal alignment with a second amino acid or nucleic acid sequence). For purposes of complementation, gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence. ) and then the amino acid residue or nucleotide at the corresponding amino acid position or nucleotide position. The amino acid residues at positions in the first sequence are compared to the corresponding positions in the second sequence. or nucleotide, the molecules are identical at that position. The percent identity between sequences is a function of the number of identical positions shared by these sequences. (i.e., % identity = number of identical overlapping positions / total number of positions x 100%). In some embodiments, the percent identity is calculated based on the sequence identity of the two sequences. The amino acid sequence or the nucleotide sequence is determined over the entire length.

[0168] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be calculated mathematically. This can also be achieved using an algorithm. A mathematical algorithm is used to compare two sequences. Preferred, non-limiting examples of rhythms are those described in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873 5877) modified from Karlin and Altschul (1 990, Proc. Natl. Acad. Sci. USA 87:2264 2268). The algorithms used were NBLAST and XBLAST from Altschul et al. (1990, J. Mol. Biol. 215:403). The BLAST nucleotide search is performed using a BLAST search engine with a score of 100 and a word count of 100. The NBLAST nucleotide program parameters were set to nucleotide length = 12, and the Nucleotide sequences homologous to the nucleic acid molecules described herein can be obtained by BLAST protein searches. Searches were performed using the XBLAST program parameters set to, for example, score 50 and word length = 3. This can be performed to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST was used as described by Altschul et al. It can be used as described in the literature (1997, Nucleic Acids Res. 25:3389 3402). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules. (Ibid.) BLAST, Gapped BLAST, and PSI Blast programs can be used. When using the default parameters of each program (e.g., XBLAST and NBLAST), can be used (e.g., on the World Wide Web, at ncbi.nlm.nih.gov) (See National Center for Biotechnology Information (NCBI) for sequence comparison.) Another preferred, non-limiting example of a mathematical algorithm is that described by Myers and Miller (1988). CABIOS 4:11 17). Such an algorithm is useful for GCG sequence alignment. It is incorporated into the ALIGN program (version 2.0), which is part of the ALIGN software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residues are Table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0169] The percent identity between two sequences can be determined using the techniques described above, with or without allowing gaps. When determining percent identity, the positive Only exact matches are counted.

[0170] In certain embodiments, provided herein are KIT polypeptides (e.g., human KIT (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to the D4 region of the sequence VH CDR1, VH CDR2, and VH CDR3, as well as H1, H2, and H3, containing the amino acid sequences of sequences 16 to 18 a VH domain containing one, two, three, or four framework regions of H1, H2, H3, H4, or H5; (see Table 5A); and / or (ii) VL CDR1, VL CDR2, VL CDR3, VL CDR4, VL CDR5, VL CDR6, VL CDR7, VL CDR8, VL CDR9, VL CDR10, VL CDR11, VL CDR12, VL CDR13, VL CDR14, VL CDR15, VL CDR16, VL CDR17, VL CDR L CDR2, and VL CDR3, and one, two, three, or four of L1, L2, L3, or L4 an antibody or antigen-binding fragment thereof comprising a VL domain including framework regions (see Table 5B); do.

[0171] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each comprise a VH CDR1 comprising the amino acid sequences of SEQ ID NOs: 16 to 18. , VH CDR2, and VH CDR3, and the framework region FR1 of H1, H2, H3, H4, or H5. In one embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) comprises a VH domain containing the VH domain. The antibodies described herein that immunospecifically bind to the amino acid sequences of SEQ ID NOS: 16-18, respectively. VH CDR1, VH CDR2, and VH CDR3, including sequences, and H1, H2, H3, H4, or H5 In one embodiment, the KIT polypeptide (e.g., and the D4 region of human KIT), respectively. VH CDR1, VH CDR2, and VH CDR3, as well as H1, H2, H3, and H4, containing the amino acid sequences of Nos. 16 to 18 In one embodiment, the VH domain comprises the framework region FR3 of KIT or H5. The antibodies described herein that immunospecifically bind to a polypeptide (e.g., the D4 region of human KIT) VH CDR1, VH CDR2, and VH CDR3 each containing the amino acid sequences of SEQ ID NOs: 16 to 18, and and a VH domain comprising the framework region FR4 of H1, H2, H3, H4, or H5.

[0172] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each comprise a VH CDR1 comprising the amino acid sequences of SEQ ID NOs: 16 to 18. , VH CDR2, and VH CDR3, and framework regions FR1 and FR2 of H1, H2, H3, H4, or H5. In one embodiment, the KIT polypeptide (e.g., the D of human KIT) comprises a VH domain comprising R2. The antibodies described herein that immunospecifically bind to the nucleotide sequences of SEQ ID NOs: 16 to 18 are also shown in Table 4. VH CDR1, VH CDR2, and VH CDR3 containing amino acid sequences, and H1, H2, H3, H4, or H5 fragments. In one embodiment, the KIT comprises a VH domain comprising framework regions FR1, FR2, and FR3. An antibody described herein that immunospecifically binds to a polypeptide (e.g., the D4 region of human KIT). VH CDR1, VH CDR2, and VH CDR3 each containing the amino acid sequences of SEQ ID NOs: 16 to 18, and a VH domain comprising framework regions FR1, FR2, FR3, and FR4 of H1, H2, H3, H4, or H5. Including the main.

[0173] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each comprise a VH CDR1 comprising the amino acid sequences of SEQ ID NOs: 16 to 18. , VH CDR2, and VH CDR3, and framework regions FR1 and FR2 of H1, H2, H3, H4, or H5. In one embodiment, the KIT polypeptide (e.g., the D of human KIT) comprises a VH domain comprising R3. The antibodies described herein that immunospecifically bind to the nucleotide sequences of SEQ ID NOs: 16 to 18 are also shown in Table 4. VH CDR1, VH CDR2, and VH CDR3 containing amino acid sequences, and H1, H2, H3, H4, or H5 fragments. It comprises a VH domain comprising framework regions FR1, FR3, and FR4.

[0174] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each comprise a VH CDR1 comprising the amino acid sequences of SEQ ID NOs: 16 to 18. , VH CDR2, and VH CDR3, and framework regions FR1 and FR2 of H1, H2, H3, H4, or H5. It contains a VH domain containing R4.

[0175] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each comprise a VH CDR1 comprising the amino acid sequences of SEQ ID NOs: 16 to 18. , VH CDR2, and VH CDR3, and framework regions FR1, FR2 of H1, H2, H3, H4, or H5 , and a VH domain comprising FR4.

[0176] In one embodiment, the human or humanized KIT polypeptide (e.g., the D4 region of human KIT) is immunized. The antibodies described herein that specifically bind to the amino acid sequences of SEQ ID NOs: 16 to 18, respectively. VH CDR1, VH CDR2, and VH CDR3, and H1, H2, H3, H4, or H5 frameworks comprising In one embodiment, the KIT polypeptide (e.g., The antibodies described herein that immunospecifically bind to the D4 region of human KIT each have the sequence VH CDR1, VH CDR2, and VH CDR3, as well as H1, H2, H3, and H4, comprising the amino acid sequences of Nos. 16 to 18. The VH domain comprises the framework regions FR2, FR3, and FR4 of H4, or H5.

[0177] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each comprise a VH CDR1 comprising the amino acid sequences of SEQ ID NOs: 16 to 18. , VH CDR2, and VH CDR3, and framework regions FR3 and FR4 of H1, H2, H3, H4, or H5. It contains a VH domain containing R4.

[0178] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. a VL CDR2, a VL CDR3, and a framework region FR1 of L1, L2, L3, or L4; In one embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) comprises an immunizing domain. The antibodies described herein that specifically bind to the amino acid sequences of SEQ ID NOs: 19 to 21, respectively. VL CDR1, VL CDR2, and VL CDR3, and framework regions of L1, L2, L3, or L4, In one embodiment, the KIT polypeptide (e.g., human KIT) comprises a VL domain comprising region FR2. The antibodies described herein that immunospecifically bind to the D4 region of SEQ ID NOs: 19 to 21, respectively. VL CDR1, VL CDR2, and VL CDR3, and L1, L2, L3, or L4, each containing an amino acid sequence of In one embodiment, the KIT polypeptide ( For example, the antibodies described herein that immunospecifically bind to the D4 region of human KIT are: VL CDR1, VL CDR2, and VL CDR3 containing the amino acid sequences of SEQ ID NOs: 19 to 21, and L1, L2, L 3, or a VL domain comprising the framework region FR4 of L4.

[0179] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. , VL CDR2, and VL CDR3, and framework regions FR1 and FR2 of L1, L2, L3, or L4 In one embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) comprises a VL domain comprising the VL domain. The antibodies described herein that immunospecifically bind to the amino acid sequence of SEQ ID NOs: 19 to 21, respectively. VL CDR1, VL CDR2, and VL CDR3 containing the amino acid sequence, and the frame of L1, L2, L3, or L4 In one embodiment, the KIT polypeptide comprises a VL domain comprising framework regions FR1, FR2, and FR3. The antibodies described herein that immunospecifically bind to a peptide (e.g., the D4 region of human KIT) VL CDR1, VL CDR2, and VL CDR3 each containing the amino acid sequences of SEQ ID NOs: 19 to 21, and L and a VL domain comprising framework regions FR1, FR2, FR3, and FR4 of L1, L2, L3, or L4. nothing.

[0180] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. , VL CDR2, and VL CDR3, and framework regions FR1 and FR3 of L1, L2, L3, or L4 In one embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) comprises a VL domain comprising the VL domain. The antibodies described herein that immunospecifically bind to the amino acid sequence of SEQ ID NOs: 19 to 21, respectively. VL CDR1, VL CDR2, and VL CDR3 containing the amino acid sequence, and the frame of L1, L2, L3, or L4 It contains a VL domain that includes framework regions FR1, FR3, and FR4.

[0181] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. , VL CDR2, and VL CDR3, and framework regions FR1 and FR4 of L1, L2, L3, or L4 It comprises a VL domain containing

[0182] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. , VL CDR2, and VL CDR3, and the framework regions FR1, FR2, and FR3 of L1, L2, L3, or L4. and a VL domain comprising FR4.

[0183] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. , VL CDR2, and VL CDR3, and framework regions FR2 and FR3 of L1, L2, L3, or L4 In one embodiment, the KIT polypeptide (e.g., the D4 region of human KIT) comprises a VL domain comprising the VL domain. The antibodies described herein that immunospecifically bind to the amino acid sequence of SEQ ID NOs: 19 to 21, respectively. VL CDR1, VL CDR2, and VL CDR3 containing the amino acid sequence, and the frame of L1, L2, L3, or L4 It contains a VL domain that includes framework regions FR2, FR3, and FR4.

[0184] In one embodiment, a polypeptide that immunospecifically binds to a KIT polypeptide (e.g., the D4 region of human KIT) The antibodies described herein each have a VL CDR1 comprising the amino acid sequences of SEQ ID NOs: 19 to 21. , VL CDR2, and VL CDR3, and framework regions FR3 and FR4 of L1, L2, L3, or L4 It comprises a VL domain containing

[0185] In certain embodiments, provided herein are KIT polypeptides (e.g., human KIT (i) an antibody or antigen-binding fragment thereof that immunospecifically binds to the D4 region of the sequence VH CDR1, VH CDR2, and VH CDR3 containing the amino acid sequences of sequences 16 to 18, and VH domain H a VH domain comprising any one of framework regions FR1 to FR4 of H257 to HH281 (see Table 5C); and (ii) VL CDR1, VL CDR2, and VL CDR3 each comprising the amino acid sequences of SEQ ID NOs: 19 to 21. a VL domain comprising DR3 and FR1 to FR4 of any one of VL domains LL65 to LL76 (see Table 5D); and wherein the antibody or antigen-binding fragment thereof comprises:

[0186] In certain embodiments, provided herein are KIT polypeptides (e.g., human KIT an antibody or antigen-binding fragment thereof that immunospecifically binds to: (i) a polypeptide shown in Table 2 or 3; VH CDR1, VH CDR2, and VH CDR3, and VH domain H, comprising a combination of amino acid sequences as described above. 1 to H5 (see Table 5A) and HH257 to HH281 (see Table 5C) and (ii) a VH domain comprising a VH domain selected from the group consisting of: ... VL CDR1, VL CDR2, and VL CDR3 comprising a combination of amino acid sequences shown in either of the above (VL CDR1, VL CDR2, and VL CDR3) and FR1 to FR4 of any one of VL domains L1 to L4 (see Table 5B) and LL65 to LL76 (see Table 5D). The antibody or antigen-binding fragment thereof comprises a VL domain comprising:

[0187] In certain embodiments, provided herein are KIT polypeptides (e.g., human KIT an antibody or antigen-binding fragment thereof that immunospecifically binds to: (i) a polypeptide shown in Table 2 or 3; and a VH CDR1, a VH CDR2, and a VH CDR3 comprising a combination of amino acid sequences as shown in, for example, FIG. The corresponding framework regions comprising the sequences flanking the VH CDRs shown in any one of A to 3I. and (ii) a VH domain comprising regions FR1 to FR4; and (iii) a VH domain comprising regions FR1 to FR4; and (iv) a VH domain comprising regions FR1 to FR4; VL CDR1, VL CDR2, VL CDR3, VL CDR4, VL CDR5, VL CDR6, VL CDR7, VL CDR8, VL CDR9, VL CDR10, VL CDR11, VL CDR12, VL CDR13, VL CDR14, VL CDR15, VL CDR16, VL CDR17, VL CDR18, VL CDR19, VL C 2, and VL CDR3, and sequences adjacent to the VL CDRs shown, for example, in any one of Figures 3A-3I. an antibody or its antigen-binding domain comprising a VL domain comprising corresponding framework regions FR1 to FR4 containing the sequence; It is the original binding fragment.

[0188] Table 5A: VH domain framework regions (FR) [Table 5]

[0189] Table 5B: VL domain framework regions (FR) [Table 6]

[0190] Table 5C: Framework region sequences of VH domains HH257 to HH281 [Table 7]

[0191] Table 5D: Framework region sequences of VL domains LL65 to LL76 [Table 8]

[0192] In certain embodiments, provided herein are KIT polypeptides (e.g., human KIT an antibody (e.g., a human or humanized antibody) that immunospecifically binds to the D4 region of the A fragment having: (i) an amino acid sequence: [ka] (where X at Kabat position 11 H1 , Kabat position 20 X H2 , Kabat position 38 X H3 , Kabat position 67 X H4 , Kabat position 69 X H5 , Kabat position 72 X H6 , Kabat position 81 X H7 , and X at Kabat position 87 H8 teeth, and / or (ii) an amino acid sequence [ka] (where X at Kabat position 10 K1 , Kabat position 46 X K2 , Kabat position 63 X K3 , Kabat position 80 X K4 , Kabat position 85 X K5 , and X at Kabat position 87 K6 are independently selected from any amino acid an antibody (e.g., a human or humanized antibody) or an antigen-binding fragment thereof comprising a VL domain comprising In certain embodiments, the VH and / or VL domains are, for example, MHC class II. in the absence of non-human germline binders to the epitope, e.g., MHC class II As judged by the above, it is non-immunogenic.

[0193] In certain embodiments, provided herein are KIT polypeptides (e.g., human KIT (D4 region of) an antibody that immunospecifically binds to: (i) the amino acid sequence: [ka] (where X at Kabat position 11 H1 , Kabat position 20 X H2 , Kabat position 38 X H3 , Kabat position 67 X H4 , Kabat position 69 X H5 , Kabat position 72 X H6 , Kabat position 81 X H7 , and X at Kabat position 87 H8 teeth, (ii) a VH domain comprising a VH domain comprising a VH domain selected from the amino acid combinations set forth in Table 6B; and / or (ii) a VH domain comprising ... selected from the amino acid combinations set forth in Table 6B; Amino acid sequence: [ka] (where X at Kabat position 10 K1 , Kabat position 46 X K2 , Kabat position 63 X K3 , Kabat position 80 X K4 , Kabat position 85 X K5 , and X at Kabat position 87 K6 is selected from the amino acid combinations shown in Table 6A. In certain embodiments, the VH and / or VL domains are selected from the group consisting of: The VL domain may be, for example, an epitope that binds to MHC class II, e.g., an epitope that binds to MHC class II. It is non-immunogenic as determined by the absence of corresponding non-human germline binders.

[0194] In one embodiment, X at Kabat position 11 H1 is an amino acid with an aliphatic side chain (e.g., A hydrophilic side chain, or a non-polar side chain branched chain amino acid (BCAA), such as L or V. In Kabat position 20, X H2 is an amino acid with an aliphatic side chain (e.g., a hydrophobic side chain, or is a non-polar side chain branched chain amino acid (BCAA), e.g., L or V. In one embodiment, Kab at position 38 X H3 is a polar side chain (e.g., a hydrophilic side chain, a basic side chain, or a charged side chain, e.g., In one embodiment, the amino acid is an amino acid having a positively or negatively charged side chain, such as K or R. In Kabat position 67, X H4 is an amino acid with an aliphatic side chain (e.g., a hydrophobic side chain or A non-polar side chain branched chain amino acid (BCAA)), such as V or A. In one embodiment, Kabat X at position 69 H5 is an amino acid with an aliphatic side chain (e.g., a hydrophobic side chain or a non-polar side chain, branched chain) In one embodiment, X at Kabat position 72 is a BCAA, e.g., L or I. H6 teeth, An amino acid having an acidic side chain, e.g., E or D. In one embodiment, an amino acid at Kabat position 81 X H7 is an amino acid or its amide derivative having an acidic side chain, e.g., Q(uncharged / amino acid of E) In one embodiment, X at Kabat position 87 is a methyl group (a derivative) or E. H8 is an aliphatic hydroxyl The amino acid may be an amino acid having a hydrophilic side chain, such as S or T.

[0195] In one embodiment, X at Kabat position 11 H1 is an aliphatic amino acid, e.g., a branched chain amino acid (BCAA), e.g., V; X at Kabat position 20 H2 is an aliphatic amino acid, e.g., a branched chain amino acid BCAA, e.g., L; X at Kabat position 38 H3 is an amino acid with a polar side chain, e.g. For example, R; X at Kabat position 67 H4 is an amino acid with an aliphatic side chain, e.g., A; Ka X at bat position 69 H5 is an amino acid with an aliphatic side chain, e.g., L; X at Kabat position 72 H6 is an amino acid with a polar side chain, e.g., D; X at Kabat position 81 H7 is an acidic amino acid an amino acid having an amide derivative of, for example, Q; H8 is an aliphatic and amino acids with hydroxyl side chains, such as T.

[0196] In one embodiment, X at Kabat position 11 H1 is an aliphatic amino acid, e.g., a branched chain amino acid (BCAA), e.g., V; X at Kabat position 20 H2 is an aliphatic amino acid, e.g., a branched chain amino acid BCAA, e.g., V; X at Kabat position 38 H3 is an amino acid with a polar side chain, e.g. For example, R; X at Kabat position 67 H4 is an amino acid with an aliphatic side chain, e.g., A; Ka X at bat position 69 H5 is an amino acid with an aliphatic side chain, e.g., I; X at Kabat position 72 H6 is an amino acid with a polar side chain, e.g., D; X at Kabat position 81 H7is an acidic amino acid , e.g., E; X at Kabat position 87 H8 is an amino acid having an aliphatic hydroxyl side chain, For example, T.

[0197] In a specific embodiment, X at Kabat position 10 K1 is an aromatic amino acid, e.g., F, or An amino acid having an aliphatic hydroxyl side chain, e.g., S. In certain embodiments, K abat position 46 X K2 is an amino acid having an aliphatic side chain (e.g., a hydrophobic amino acid), e.g., A or an amino acid having an aliphatic hydroxyl side chain, e.g., S. In one embodiment, X in Kabat position 63 K3 is an amino acid with an aliphatic hydroxyl side chain, e.g., T or S In a specific embodiment, X at Kabat position 80 K4 has an aliphatic hydroxyl side chain In one embodiment, the amino acid is a carboxylic acid, such as S, or an aromatic amino acid, such as P. , Kabat position 85 X K5 is an acidic amino acid, e.g., D, or an aliphatic hydroxyl side chain. In one embodiment, X at Kabat position 87 is an amino acid that is K6 is an aromatic amino acid The acid is, for example, F or Y.

[0198] In a specific embodiment, X at Kabat position 10 K1 is an aliphatic hydroxyl side chain amino acids, e.g., S; X at Kabat position 46 K2 is an amino acid with an aliphatic side chain (e.g., hydrophobic amino acid), e.g., A; X at Kabat position 63 K3 has an aliphatic hydroxyl side chain an amino acid, e.g., T; X at Kabat position 80 K4 is an aromatic amino acid, e.g., P X in Kabat position 85 K5 is an acidic amino acid, e.g., D; X at Kabat position 87 K6 is aromatic Aromatic amino acids, for example, F.

[0199] In a specific embodiment, X at Kabat position 10 K1 is an aromatic amino acid, e.g., F; X in Kabat position 46 K2 is an amino acid having an aliphatic side chain (e.g., a hydrophobic amino acid), e.g. , A; X at Kabat position 63 K3 is an amino acid with an aliphatic hydroxyl side chain, e.g., T and X at Kabat position 80 K4 is an aliphatic hydroxyl side chain, e.g., S; Kabat position 85 X K5 is an acidic amino acid, e.g., D; X at Kabat position 87 K6 is an aromatic amino acid, e.g. If so, it is F.

[0200] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies or antigen-binding fragments thereof described herein that bind: (i) the amino acid sequence: [ka] (where X at Kabat position 11 H1 is an amino acid with an aliphatic side chain, e.g., V, and Kabat X at position 20 H2 is an amino acid with an aliphatic side chain, e.g., L, and X at Kabat position 38 H3 teeth, Amino acids with polar side chains, e.g., K and X at Kabat position 67 H4 has an aliphatic side chain an amino acid, e.g., A and X at Kabat position 69 H5 is an amino acid with an aliphatic side chain, e.g. For example, L and X at Kabat position 72 H6 is an acidic amino acid, e.g., D, and X at Kabat position 81 H7 is an acidic amino acid or an amide derivative thereof, e.g., Q, and X at Kabat position 87 H8 Is fat (ii) a VH domain comprising an amino acid having an aliphatic hydroxyl side chain, e.g., T; and Amino acid sequence [ka] (where X at Kabat position 10 K1 is an amino acid with an aliphatic hydroxyl side chain, e.g., S There is an X at Kabat position 46 K2 is an amino acid with an aliphatic side chain, e.g., A, and has a Kabat position 63X K3 is an amino acid with an aliphatic hydroxyl side chain, e.g., T, at Kabat position 80 X K4 is an aromatic amino acid, e.g., P, and X at Kabat position 85 K5 is an acidic amino acid, e.g. For example, D and X at Kabat position 87 K6 VL domains containing aromatic amino acids, e.g., F Includes

[0201] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies or antigen-binding fragments thereof described herein that bind: (i) the amino acid sequence: [ka] (where X at Kabat position 11 H1 is an amino acid with an aliphatic side chain, e.g., V, and Kabat X at position 20 H2 is an amino acid with an aliphatic side chain, e.g., V, and X at Kabat position 38 H3 teeth, Amino acids with polar side chains, e.g., R and X at Kabat position 67 H4 has an aliphatic side chain an amino acid, e.g., A and X at Kabat position 69 H5 is an amino acid with an aliphatic side chain, e.g. For example, I and X at Kabat position 72 H6 is an acidic amino acid, e.g., D, and X at Kabat position 81 H7 is an acidic amino acid, e.g., E, and X at Kabat position 87 H8 is an aliphatic hydroxyl side chain (ii) a VH domain comprising an amino acid sequence having the amino acid sequence [ka] (where X at Kabat position 10 K1 is an aromatic amino acid, e.g., F, and X at Kabat position 46 K2 teeth , an amino acid having an aliphatic side chain, e.g., A, and X at Kabat position 63 K3 is an aliphatic hydrochloride Amino acids with an xyl side chain, e.g., T and X at Kabat position 80 K4 is an aliphatic hydroxyl Amino acids with a silyl side chain, e.g., S and X at Kabat position 85 K5 is an acidic amino acid, e.g. For example, D and X at Kabat position 87 K6 VL domains containing aromatic amino acids, e.g., F Including Inn.

[0202] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies or antigen-binding fragments thereof described herein that bind to (i) SEQ ID NOs: 16 to 18, respectively and (ii) a VH domain comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of: VL domains containing numbers 7, 8, 9, or 10.

[0203] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies or antigen-binding fragments thereof described herein that bind to (i) SEQ ID NOs: 16 to 18, respectively and (ii) a VH domain comprising a VH CDR1, a VH CDR2, and a VH CDR3 comprising the amino acid sequences of: Acid sequence [ka] (where X at Kabat position 10 K1 , Kabat position 46 X K2 , Kabat position 63 X K3 , Kabat position 80 X K4 , Kabat position 85 X K5 , and X at Kabat position 87 K6 is selected from the amino acid combinations shown in Table 6A. The VL domain comprises a VL domain containing a VL domain selected from the group consisting of:

[0204] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies or antigen-binding fragments thereof described herein that bind to: (i) SEQ ID NO: 2, 3, 4, or 5 and (ii) a VH domain comprising the amino acid sequences of SEQ ID NOs: 19 to 21, respectively. The present invention comprises a VL domain comprising a VL CDR1, a VL CDR2, and a VL CDR3.

[0205] In certain embodiments, the antibodies immunospecifically target a KIT polypeptide (e.g., the D4 region of human KIT). The antibodies or antigen-binding fragments thereof described herein that bind to the IgG1A-IgG2A antigen-binding fragments have the following structure: (i) the amino acid sequence: [ka] (where X at Kabat position 11 H1 , Kabat position 20 X H2 , Kabat position 38 X H3 , Kabat position 67 X H4 , Kabat position 69 X H5 , Kabat position 72 X H6 , Kabat position 81 X H7 , and X at Kabat position 87 H8 teeth, (ii) a VH domain comprising a VH domain comprising a VH domain selected from the amino acid combinations set forth in Table 6B; and (iii) a VH domain comprising a VH domain comprising a VH domain selected from the amino acid combinations set forth in Table 6B; and (iv) a VH domain comprising a VH domain comprising a VH domain selected from the amino acid combinations set forth in Table 6B; and a VL domain comprising VL CDR1, VL CDR2, and VL CDR3 comprising the amino acid sequences of SEQ ID NOs: 19 to 21; Includes

[0206] Table 6A: VK domain amino acid substitutions [Table 9] TIFF2025060938000027.tif237170TIFF2025060938000028.tif135170

[0207] Table 6B: VH domain amino acid substitutions [Table 10] TIFF2025060938000030.tif232170TIFF2025060938000031.tif228170TIFF2025060938000032.tif227170TIFF20250609380 00033.tif231170TIFF2025060938000034.tif230170TIFF2025060938000035.tif228170TIFF2025060938000036.tif229170

[0208] In a specific embodiment, the position of the VL chain region described herein relative to the constant region (i.e. i.e., the boundary) maintains immunospecific binding to KIT (e.g., the D4 region of human KIT) (e.g., For example, substantially, e.g., at least 50%, at least 60%, at least 70%, at least one, two, three, or four (as long as the In a specific embodiment, only the amino acid positions described herein for the constant region may be varied. The locations (i.e., boundaries) of the VH chain regions described are determined by the immunoglobulins specific for KIT (e.g., the D4 region of human KIT). Specific binding is maintained (e.g., substantially, e.g., at least 50%, at least 60%). , at least 70%, at least 80%, at least 90%, at least 95% maintained) , only one, two, three, or four amino acid positions may be varied.

[0209] In specific aspects, provided herein are compounds that are described herein, e.g., A portion comprising a VH CDR and / or a VL CDR as shown in Tables 1 to 3 and 10 to 15, wherein the VH The CDRs and VL CDRs are arranged in a spatial orientation that confers specific binding to the D4 region of human KIT. do.

[0210] In a specific embodiment, provided herein are antibodies comprising the amino acid sequences of SEQ ID NOs: 16-18. VH CDRs comprising the amino acid sequences of SEQ ID NOs: 19 to 21, and VL CDRs comprising the amino acid sequences of SEQ ID NOs: 56, 62, and 63. VH CDRs containing the amino acid sequences of SEQ ID NOs: 59 to 61, and VL CDRs containing the amino acid sequences of SEQ ID NOs: 59 to 61. VH CDRs comprising the amino acid sequences of SEQ ID NOs: 70 to 72 and VL CDRs comprising the amino acid sequences of SEQ ID NOs: 66 to 68. wherein the VH CDR and VL CDR confer specific binding to the D4 region of human KIT. In certain embodiments, the moieties are arranged in a spatial orientation that corresponds to the antibody or its antigen. In certain embodiments, the portion is a protein, e.g., a fragment containing an Fc region. It is a fusion protein containing

[0211] In specific embodiments, provided herein are VH CDRs and VH CDRs selected from Tables 13-15. and / or a portion comprising a VH CDR and a VL CDR selected from Tables 10 to 12, wherein the VH CDR and the VL CDR are arranged in a spatial orientation that confers specific binding to the D4 region of human KIT. In aspects, the moiety is an antibody or an antigen-binding fragment thereof. The moiety is a protein, for example a fusion protein that includes an Fc region.

[0212] In specific embodiments, provided herein are antibodies selected from those presented in Tables 10-15. a portion, e.g., an antibody or antigen-binding fragment thereof, comprising VH CDRs 1-3 and VL CDRs 1-3 selected from the group consisting of: wherein the VH and VL CDRs comprise a vacancy that confers specific binding to the D4 region of human KIT. In certain embodiments, the portions described herein are arranged in an inter- direction. The VH CDRs 1-3 and / or VL CDRs 1-3 are linked in a spatial orientation that confers specific binding to the four domains. Linkers include, for example, peptide linkers.

[0213] In certain embodiments, the moieties described herein confer specific binding to the D4 region of human KIT. a linker, e.g., a peptide linker, which links the VH and / or VL CDRs in a given spatial orientation; Including cars.

[0214] In some embodiments, provided herein are antibodies selected from those presented in Tables 10-15. and an antibody or antigen-binding fragment thereof comprising VL CDRs 1 to 3 and VH CDRs 1 to 3, The antibody or antigen-binding fragment thereof immunospecifically binds to the D4 region of KIT, eg, human KIT.

[0215] In specific embodiments, the "X" amino acid of a CDR in any one of Tables 10-15 is: It is any naturally occurring amino acid that maintains specific binding affinity to the D4 region of human KIT. In one embodiment, the "X" amino acid of a CDR in any one of Tables 10-15 is a CDR of a human KIT and unnatural amino acids that maintain specific binding affinity to the D4 region of In any one of Tables 10 to 15, the "X" amino acid of the CDR is an amino acid of SEQ ID NO: 16 to 21. a conservative substitution of the corresponding amino acids in the CDRs with the amino acid sequence of the D4 region of human KIT; Specific binding affinity for the region is maintained.

[0216] In specific embodiments, the "X" amino acid of a CDR in any one of Tables 10-15 is: In certain embodiments, the amino acids A, G, T, K, or L are selected from the group consisting of any of Tables 10-15. The "X" amino acid of any one of the CDRs is the amino acid A, G, T, Y, C, or S. In some aspects of the embodiment, the specific binding affinity for the D4 region of human KIT is maintained. do.

[0217] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are those provided in Tables 10-15. It comprises a VH CDR and / or a VL CDR selected from those shown.

[0218] In certain embodiments, the CDRs, e.g., VL CDRs 1-3 and VH CDR 1 shown in Tables 10-15, are Any one of the above three amino acids contains one or more (e.g., two, three, four, or five) "X" amino acids. , where each "X" amino acid is a specific amino acid of an antibody or fragment thereof against the D4 region of human KIT. It can be any amino acid that is capable of maintaining binding. Table 10: VL CDR1 [Table 11] Table 11: VL CDR2 [Table 12] Table 12: VL CDR3 [Table 13] Table 13: VH CDR1 [Table 14] Table 14: VH CDR2 [Table 15] Table 15: VH CDR3 [Table 16]

[0219] In some embodiments, provided herein are antibodies selected from those presented in Tables 20-23. and / or VH FRs 1-4 selected from those presented in Tables 16-19. or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to KIT, e.g. , which immunospecifically binds to the D4 region of human KIT.

[0220] In specific embodiments, the "X" amino acid of an FR in any one of Tables 16-23 is a human The amino acid sequence may be any naturally occurring amino acid that maintains specific binding affinity for the D4 region of human KIT. In certain embodiments, the "X" amino acid of the CDRs in any one of Tables 16-23 is a CDR of human KIT. In particular embodiments, the amino acid is an unnatural amino acid that maintains specific binding affinity for the D4 region. In this case, the "X" amino acid of the CDR in any one of Tables 16 to 23 is an amino acid of SEQ ID NOs: 16 to 21. a conservative substitution of the corresponding amino acids of the CDRs with the amino acid sequence of the D4 region of human KIT; The specific binding affinity for the antibody is maintained.

[0221] In specific embodiments, the "X" amino acid of a CDR in any one of Tables 16-23 is: The amino acids are A, G, T, K, or L. In certain embodiments, the amino acids in any one of Tables 16-23 are The "X" amino acids of the CDRs in this embodiment are the amino acids A, G, T, Y, C, or S. In certain embodiments, the specific binding affinity for the D4 region of human KIT is maintained. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are selected from the group consisting of antibodies, antibodies, and antigen-binding fragments thereof provided in Tables 16-23. It comprises a VH CDR and / or a VL CDR selected from those shown.

[0222] In certain embodiments, FRs, e.g., VL FRs 1-4 and VH FRs 1-4 shown in Tables 16-23, any one of which contains one or more (e.g., two, three, four, or five) "X" amino acids; where each "X" amino acid is a specific binding site for the antibody or fragment thereof to the D4 region of human KIT. The amino acid may be any amino acid that can maintain the desired binding affinity. Table 16: VH FR1 [Table 17] Table 17: VH FR2 [Table 18] Table 18: VH FR3 [Table 19] Table 19: VH FR4 [Table 20] Table 20: VL FR1 [Table 21] Table 21: VL FR2 [Table 22] Table 22: VL FR3 [Table 23] Table 23: VL FR4 [Table 24]

[0223] In one aspect, the antibodies described herein comprise one or more amino acid deletions, additions, and / or modifications. It contains an Fc region containing a decoration.

[0224] As used herein, "Fc region" refers to the portion of an antibody that excludes the first constant region immunoglobulin domain. The polypeptides containing the constant region of the human IgA, IgD, and IgG are therefore Constant region immunoglobulin domains, the last three constant region immunoglobulin domains of IgE and IgM The term refers to the main domains and the flexible hinges at the N-termini of these domains. For IgG, the Fc region may include the immunoglobulin domain C. gamma 2 and C gamma 3 (Cγ2 and Cγ3) and the hindrance between C gamma 1 (Cγ1) and C gamma 2 (Cγ2) Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region typically contains its carbohydrate The hydroxyl terminus contains residues C226 or P230, where the numbering is as in Kabat et al. (1991, NIH Publication ication 91-3242, National Technical Information Service, Springfield, VA The EU index as presented in Kabat's paper is " refers to the residue numbering of the human IgG1 EU antibody as described in Kabat et al. (supra). In some embodiments, the Fc region comprises a non-naturally occurring Fc region. at one or more Fc positions, including but not limited to, Kabat 270, 272, 312, 315, 356, and 358 exist.

[0225] In one embodiment, provided herein are equivalent antibodies (e.g., antibodies having a wild-type Fc region). Fc ligands (e.g., antibodies having the same amino acid sequence except that the Fc receptor the D4 region of KIT, which contains an Fc region with altered binding properties to receptors, e.g., C1q. The antibody or antigen-binding fragment thereof described herein specifically binds to the antibody.

[0226] The affinity and binding properties of the Fc region for its ligands are determined by Fc-FcγR interactions, i.e., Various in vitro assays known in the art can be used to determine the specific binding of an Fc region to an FcγR. The assay can be determined by a biochemical or immunological assay. Methods include, but are not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), or kinetics (e.g., BIACORE® analysis), as well as other methods methods, such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), These include gel electrophoresis, and chromatography (e.g., gel filtration). Other methods include, but are not limited to, utilizing labels on one or more of the components under consideration and / or A variety of detection methods may be used, including chromogenic, fluorescent, luminescent, or isotopic labels. can.

[0227] In one embodiment, the Fc region of an antibody described herein has one or more In a specific embodiment, the antibodies described herein have enhanced binding to Fc ligands. The Fc regions of the described antibodies have enhanced binding to Fc receptors. In such a manner, the Fc region of the antibody described herein has enhanced binding to the Fc receptor FcγRIIIA. In one specific embodiment, the Fc region of the antibody described herein has Fc In another specific embodiment, the antibodies described herein have enhanced binding to the receptor FcRn. The Fc region of the antibody described in has enhanced binding to C1q compared to comparable molecules.

[0228] In some embodiments, the serum half-life of a protein comprising an Fc region is determined by the binding of the Fc region to FcRn. In one embodiment, the present invention provides a method for increasing the binding affinity of a target protein. The Fc regions of the antibodies described herein have improved serum half-lives compared to comparable molecules.

[0229] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the use of specific cytotoxic cells (e.g. Fc receptors (FcRs) present on immune cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) These cytotoxic cells specifically bind to antigen-bearing target cells, A form of cytotoxicity that allows the target cells to be subsequently killed by a cytotoxin Specific high-affinity IgG antibodies directed against the surface of target cells are essential for such killing. Lysis of the target cell involves direct cell-to-cell contact and is a complement In addition to antibodies, Fc regions have the ability to specifically bind to antigen-bearing target cells. Other proteins containing the Fc region, specifically Fc fusion proteins, have been shown to induce cell-mediated cytotoxicity. For simplicity, we will assume that the activity of the Fc fusion protein Cell-mediated cytotoxicity is also referred to herein as ADCC activity.

[0230] Any particular protein, e.g., an antibody, containing an Fc region can mediate lysis of target cells by ADCC. To assess the ADCC activity of an Fc region, the Fc region can be assayed for its ability to mediate ADCC. The target cell-bound antibody, comprising: These are added to the target cells in combination with immune effector cells that can cause cell lysis. Typically, labels (e.g., radioactive substrates, fluorescent dyes, or natural intracellular tags) are removed from lysed cells. Useful effectors for such assays are: Cells include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985 79:277-282; Bruggemann et al. Wilkinson et al., 1987, J Exp. Med. 166:1351-1361; Wilkinson et al., 2001, J Immunol. Met methods 258:183-191; Patel et al., 1995 J Immunol. Methods 184:29-38 Alternatively, or in addition, the ADCC activity of a protein comprising an Fc region can be measured in vivo, e.g., by , animal models, e.g., Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-65 The present invention can be evaluated in animal models disclosed in .

[0231] In one embodiment, a protein comprising an Fc region, such as an antibody described herein, In another specific embodiment, the antibody has enhanced ADCC activity compared to other proteins such as antibodies. In embodiments, the antibodies described herein that comprise an Fc region have a higher affinity for Fc receptors than comparable antibodies. They have enhanced binding to FcγRIIIA and enhanced ADCC activity. In some embodiments, the antibodies described herein that comprise an Fc region have enhanced immunoglobulin activity compared to a comparable antibody. It has both enhanced ADCC activity and improved serum half-life.

[0232] "Complement dependent cytotoxicity" and "CDC" refer to the lysing of a target cell in the presence of complement. The complement activation pathway involves the formation of a complex of the first component of the complement system (C1q) with a molecule, e.g., a cognate antigen. Complement activation is initiated by binding to the antibody produced. CDC assay as described in O-Santoro et al., 1996, J. Immunol. Methods, 202:163 In one embodiment, the antibodies described herein that comprise an Fc region can be In another embodiment, the antibody has an Fc region and has enhanced CDC activity compared to a comparable antibody. The antibodies described herein, including those described herein, have enhanced CDC activity and improved serum seroconversion compared to comparable antibodies. Both have half-lives.

[0233] In one embodiment, the antibodies described herein are selected from the EU index as set forth in the Kabat reference. 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334 (e.g., 1 Amino acid modifications (e.g., substitutions, deletions, or additions at one, two, three, or four positions) Optionally, the Fc region comprises an Fc region comprising a non-naturally occurring amino acid residue (e.g., a non-naturally occurring amino acid residue). and / or amino acid modifications (e.g., substitutions, deletions, or additions) or non- Naturally occurring amino acid residues may be included (see, e.g., U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,0 56; PCT Patent Publication Nos. WO 01 / 58957; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; and See WO 05 / 040217, each of which fully, but in particular, describes such modifications. (The disclosure of which is incorporated herein by reference.) In a further embodiment, one or more of the Fc regions The functionality of the amino acid sequence can be enhanced by amino acid modifications (e.g., substitutions, deletions, or additions) or non-naturally occurring modifications at one or more positions. The Fc region comprises amino acid residues substantially identical to, e.g., at least 5 0%, at least 60%, at least 70%, at least 80%, at least 90%, at least 9 For example, such Fc functions include effector functions, e.g., CDC. Alternatively, ADCC, or binding affinity to Fc receptors may be mentioned. In one or more positions, an amino acid modification (e.g., substitution, deletion, or addition) or a non-natural amino acid The Fc region comprising the amino acid residues exhibits at least one or more enhanced Fc activity, e.g., improved These antibodies may exhibit increased half-life, or enhanced effector function, such as ADCC or CDC. In certain embodiments, an amino acid modification (e.g., substitution, deletion, or addition) at one or more positions is ) or unnatural amino acid residues, the Fc region may have reduced Fc activity, e.g., reduced stability / half-life. They exhibit reduced life, or reduced effector function, such as ADCC or CDC.

[0234] In a specific embodiment, the antibodies described herein comprise an Fc region, wherein the Fc region The regions are numbered according to the EU index given in Kabat's reference: 234D, 234E, 234N, 2 34Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235 N, 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, 262I, 262A, 26 2T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y , 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 29 6T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L , 299A, 299S, 299V, 299H, 299F, 299E, 313F, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 32 8F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L , 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, At least one (e.g., one, two) selected from the group consisting of 332T, 332H, 332Y, and 332A , 3, or 4) amino acid modifications (e.g., substitutions, deletions, or additions) or at least Optionally, the F The c region may contain additional and / or alternative non-natural amino acid residues known to those of skill in the art (e.g. See, for example, U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publication WO 01 / 58957. (See WO 04 / 016750; WO 04 / 029207; WO 04 / 035752, and WO 05 / 040217) .

[0235] In certain embodiments, provided herein is an antibody comprising an Fc region, wherein the The Fc region is numbered 239, 330, and 331 according to the EU index as given in Kabat. 32. In a specific embodiment, provided herein is an antibody comprising an Fc region, wherein The Fc region is numbered according to the EU index as set forth in Kabat, 239D, 330L. and 332E. The Fc region is further numbered according to the EU index as set forth in Kabat et al., 252; 254, and 256. In a specific embodiment, provided herein is an antibody comprising an Fc region, wherein the Fc region is numbered according to the EU index as set forth in the Kabat reference, 239D 330L, and 332E; At least one unnatural amino acid at the above positions is selected from the EU index as set forth in Kabat. In one embodiment, the amino acid sequence is selected from the group consisting of 252Y, 254T, and 256E, numbered by In this case, an Fc region comprising such a sequence may exhibit one or more Fc activities, such as binding to an Fc receptor. In particular embodiments, the antibody exhibits affinity, or effector function, such as ADCC or CDC. In this case, an Fc region containing such a sequence has reduced Fc activity, for example, reduced Fc receptor activity. They exhibit reduced binding affinity or reduced effector function, such as ADCC or CDC. In certain embodiments, an Fc region comprising such a sequence exhibits enhanced Fc activity, e.g., Improved half-life, enhanced binding affinity to Fc receptors, or enhanced effectors - function, e.g., ADCC or CDC.

[0236] Non-limiting examples of Fc region modifications include those described in Ghetie et al., 1997, Nat Biotech. 15:637-40; Dunc an et al., 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-26 62; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantat ion 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-1199 84; Jefferis et al., 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett 54:101-104; Lund et al., 1996 , J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idu Sogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164 :1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferi s et al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6, No. 277,375; No. 5,869,046; No. 6,121,022; No. 5,624,821; No. 5,648,260; No. 6,528,624; No. 6,194,551; No. 6,737,056; No. 6,821,505; No. 6,277,375; No. 8,163,882; No. 7,355,00 No. 8; No. 7,960,512; No. 8,039,592; No. 8,039,359; No. 8,101,720; No. 7,214,775; No. 7,68 2,610; 7,741,442; U.S. Patent Publication No. 2004 / 0002587 and PCT Publication No. WO 94 / 29351; WO 99 / 5 No. 8572; No. WO 00 / 42072; No. WO 04 / 029207; No. WO 04 / 099249; No. WO 04 / 063351 do.

[0237] In certain embodiments, the antibodies described herein comprise an IgG1 Fc region, e.g., a human IgG1 In some embodiments, the Fc region may comprise one or more (e.g., one, two, three, or four) modifications. In this regard, the antibodies described herein may comprise one or more antibodies directed against an IgG2 Fc region, e.g., a human IgG2 Fc region. In one embodiment, the amino acid sequence described herein includes one, two, three, or four of the above modifications. The antibody may have one or more (e.g., one, two, or more) Fc domains directed against an IgG4 Fc region, e.g., a human IgG4 Fc region. Contains modifications of the following:

[0238] In some embodiments, the antibodies described herein comprise an Fc region, wherein the Fc region may have one or more (e.g., one, two, three, or four) glycoform modifications (e.g., removal or addition of one or more glycoforms). or substitutions), e.g., engineered glycoforms, i.e., carbohydrates covalently attached to molecules comprising an Fc region. The engineered glycoforms may have, but are not limited to, enhanced or decreased effector function. The modified glycoforms can be useful for a variety of purposes, including the following: by, for example, using modified or mutant expression strains, For example, by co-expression with DI N-acetylglucosaminyltransferase III (GnTI11) Therefore, it is possible to express molecules containing Fc regions in various organisms or cell lines derived from various organisms. or by expressing a molecule containing an Fc region and then modifying the carbohydrate(s). Methods for producing modified glycoforms are known in the art. , including Umana et al., 1999, Nat. Biotechnol 17:176-180; Davies et al. , 20017 Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:267 33-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), U.S. Pat. No. 6,602,688 4; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; PCT WO 00 / 61739A1; PC T WO 01 / 292246A1; PCT WO 02 / 311140A1; PCT WO 02 / 30954A1; WO 00061739; US 200 and Okazaki et al., 2004, JMB, 336: 1239-49. These include, but are not limited to, generating modified glycoforms of the antibodies described herein. Methods have been described in the art, including U.S. Pat. No. 7,517,670; U.S. Pat. No. 8,022,499; No. 1,856; U.S. Patent No. 8,080,415; U.S. Patent No. 8,084,222; U.S. Patent No. 7,700,321; and U.S. Patent No. Examples of suitable methods include, but are not limited to, those described in US Pat. No. 8,071,336.

[0239] In certain embodiments, the glycosylation of the Fc region is modified to increase effector function. Thus, in one embodiment, the antibodies described herein In another embodiment, the Fc region of the antibody comprises modified glycosylation of amino acid residues. Modified glycosylation of amino acid residues may result in reduced effector function, e.g., ADCC or CDC. In another embodiment, the modified glycosylation of the amino acid residues results in In some embodiments, the antibodies provided herein result in increased vector function. Modification of the glycosylation pattern of the host to enhance ADCC and CDC effector function (e.g., Sh ields et al. (2002) JBC. 277:26733; Shinkawa et al. (2003) JBC. 278:3466, and Ok (See, e.g., Azaki et al. (2004) J. Mol. Biol., 336: 1239). In this context, such modified glycosylation is similar to the glycosylation of the Fc region found naturally in vivo. Such modified glycosylation differs from or is a result of the non-natural glycosylation of the Fc region. For example, modified glycosylation of the Fc region involves modifying amino acids. or by modifying the protein / antibody with glycosyl groups different from those of its unmodified parent cell. This can be achieved by expression in cells that have been engineered to contain the activating machinery. In this regard, such cells may be characterized as those that do not express certain glycosylation enzymes or that do not express the parental glycosylation enzymes. The cells can be engineered to express specific glycosylation enzymes that are not present in the cells.

[0240] Methods for generating non-native Fc regions are known in the art. For example, amino acid substitutions and / or deletions can be made by, but not limited to, site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis (Higuchi, "PCR Protocols" PCR Protocols: A Guide to Methods and Applications, A Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (Wells et al. Gene 34:315-323 (1985)). Site-directed mutagenesis can be performed by overlap-extension PCR (Higuchi et al. See the literature, "PCR Technology: Principles and Applications of DNA Amplification" "S for DNA Amplification," Stockton Press, New York, pp. 61-70 (1989). Any desired mutation(s) can be introduced using the technique of overlap-extension PCR (Higuchi, supra). Other methods useful for generating Fc region modifications include the introduction of These methods are well known in the art (e.g., U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; No. 6,165,745; No. 6,277,375; No. 5,869,046; No. 6,121,022; No. 5,624,821; No. 5,648,26 No. 0; No. 6,528,624; No. 6,194,551; No. 6,737,056; No. 6,821,505; No. 6,277,375; Patent Publication No. 2004 / 0002587, and PCT Publication Nos. WO 94 / 29351; WO 99 / 58572; WO 00 / 42072; WO (See WO 02 / 060919; WO 04 / 029207; WO 04 / 099249; WO 04 / 063351).

[0241] In a specific embodiment, the Fc region has reduced fucosylation. In the present invention, the Fc region is afucosylated (see, e.g., U.S. Patent Application Publication No. 2005 / 0226 (See issue 867).

[0242] In certain embodiments, the antibodies described herein are non-fucosylated antibodies, e.g., the antibodies The Fc region of the human body contains sugar chains containing fucose, e.g., fucosyltransferase linked to N-acetylglucosamine. (See, e.g., U.S. Patent Nos. 7,214,775; 7,682,610; and 7,741,442. Methods for producing non-fucosylated antibodies are known in the art, for example, see U.S. Pat. For example, non-fucosylated antibodies can be produced by inducing the production of antibodies in modified host cells. The present invention can be made using a vesicle, as described, for example, in U.S. Patent Nos. 6,946,292; 7,425,446; 8,06 See, e.g., Nos. 7,232; 7,846,725; and 7,393,683. Knockout animals for this purpose have also been described, see, e.g., U.S. Pat. No. 7,737,325. In certain embodiments, the antibodies described herein that specifically bind to the D4 region of human KIT are Non-fucosylated antibodies of the present invention exhibit increased ADCC.

[0243] In certain embodiments, the antibodies described herein have a fucose content of 10 compared to that of a reference antibody. 0%, e.g., less than 65% fucose content (see, e.g., U.S. Pat. No. 7,931,895 and (See US Pat. No. 7,846,434.) In certain embodiments, the antibodies described herein are fucoproteins. characterized by a high N-linked oligosaccharide content, e.g., ... At least about 60% of the N-linked oligosaccharides in the saccharide do not contain fucose. In the present invention, N-linked oligosaccharides that do not contain fucose, such as N-linked oligosaccharides in the CH2-derived domain, The percentage of conjugated oligosaccharides is at least about 65%, about 70%, about 75%, about 80%, about 85% , about 90%, about 95% or more, and altered effector function, e.g., In certain embodiments, the D4 region of human KIT exhibits altered or reduced ADCC. The antibodies described herein that specifically bind to IL-14 have a fucose content of less than 65% and are effective against increased AD. Indicates CC.

[0244] In certain embodiments, the antibodies described herein are modified antibodies that exhibit reduced binding to FcγRs. an Fc region having altered FcγR binding activity, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, ​​388, 389, 414, Any one or more of 416, 419, 434, 435, 437, 438, or 439 (e.g., one, two, three) , or 4), wherein the numbering of residues in the Fc region is in accordance with Kabat. EU index numbering as seen in (see, e.g., U.S. Patent No. 7,183,387 For example, the antibodies described herein that include an Fc region may have reduced binding to FcγRI. and any one or more of amino acid positions 238, 265, 269, 270, 327, or 329 of the Fc region. and a number (one, two, three, or four) of amino acid modifications, where the numbering of residues in the Fc region is , the EU index numbering as found in Kabat's reference.

[0245] In certain embodiments, the antibodies described herein comprising an Fc region have a low affinity for FcγRII. and the Fc region amino acid positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438, or 439 any one or more (e.g., one, two, three, or four) amino acid modifications, The numbering of residues in the Fc region is that of the EU index as in Kabat. do.

[0246] In a specific embodiment, the antibodies described herein comprising an Fc region comprise an FcγRIII-specific antibody. and the Fc region exhibits reduced binding and is expressed at amino acid positions 238, 239, 248, 249, 252, 254, 265, 268 , 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340 , 373, 376, 382, ​​388, 389, 416, 434, 435, or 437 (e.g., 1 and (one, two, three, or four) amino acid modifications in the Fc region, wherein the numbering of residues in the Fc region is This is the EU index numbering as seen in the bat literature.

[0247] In some embodiments, a protein comprising an Fc region, such as a serum half of an antibody described herein, The reduction life is increased by increasing the binding affinity of the Fc region to FcRn.

[0248] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are EC50 of approximately 50 nM or less when measured by 50 (half-maximal effective concentration) value, KIT polypeptide (e.g., human It specifically binds to the D4 region of human KIT.

[0249] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are CHO-WT-KIT Measured by FACs using cells (CHO cells engineered to recombinantly express wild-type human KIT) When the EC 50 values ​​specific to the KIT polypeptide (e.g., the D4 region of human KIT) actively binds.

[0250] In certain embodiments, a KIT polypeptide (e.g., the D4 region of human KIT) is specifically bound to the KIT polypeptide. The antibodies or antigen-binding fragments thereof described herein have an IC of about 600 pM or less. 50 (50% inhibitory concentration) At this level, KIT phosphorylation can be blocked.

[0251] In certain embodiments, a KIT polypeptide (e.g., the D4 region of human KIT) is specifically bound to the KIT polypeptide. The antibodies or antigen-binding fragments thereof described herein have a C In certain embodiments, the KIT polypeptide (e.g., human KIT) is recombinantly expressed in HO cells. The antibodies or antigen-binding fragments thereof described herein that specifically bind to the D4 region of T are at least It is also recombinantly expressed in CHO cells at an average titer of 1.0 μg / mL.

[0252] In a specific embodiment, a KIT polypeptide (e.g., the D4 region of human KIT) is specifically bound to the KIT polypeptide. The antibodies or antigen-binding fragments thereof described herein comprise a VH domain that is non-immunogenic and VL domain, e.g., the VH domain and VL domain contain a T cell epitope. do not have.

[0253] In certain embodiments, the antibodies (or antigen-binding fragments thereof) described herein bind to the KIT cells. It does not bind to an extracellular ligand binding site, such as the SCF binding site of KIT. Thus, the antibodies (or antigen-binding fragments thereof) described herein inhibit ligand binding to KIT. For example, it does not inhibit KIT ligand (eg, SCF) binding to KIT.

[0254] In a specific embodiment, the antibodies (e.g., human or humanized antibodies) described herein inhibit Antibodies, i.e., antibodies that inhibit (e.g., partially inhibit) KIT activity, i.e., one or more KIT activities In a specific embodiment, partial inhibition of KIT activity is, for example, about 25% to about 30%. In a specific embodiment, partial inhibition of KIT activity results in about 65% or 75% inhibition. For example, about 35% to about 85% or 95% inhibition occurs. Non-limiting examples of KIT activity include K KIT dimerization, KIT phosphorylation (e.g., tyrosine phosphorylation), and downstream signaling of KIT (e.g., Induction of gene transcription (e.g., c-Myc) In some embodiments, the therapeutic effect may be enhanced by the induction or enhancement of cell proliferation, cell survival, or cell proliferation. In the present invention, the antibodies described herein inhibit KIT phosphorylation (e.g., ligand-induced phosphorylation). In a specific embodiment, the antibodies described herein inhibit the KIT cytoplasmic domain. In another specific embodiment, the method described herein inhibits KIT tyrosine phosphorylation. In yet another specific embodiment, the antibodies described herein inhibit cell proliferation. In a specific embodiment, the antibodies described herein inhibit apoE. In another specific embodiment, the antibodies described herein induce cell division. Induce cell differentiation in cells expressing, for example, KIT, e.g., human KIT. In certain embodiments, the antibodies described herein inhibit KIT activity but do not inhibit KIT dimerization. In another specific embodiment, the antibodies described herein do not inhibit KIT activity. However, it does not inhibit ligand binding to KIT, and for example, it inhibits the binding of KIT ligands (e.g., SCF) binding but inhibits KIT dimerization.

[0255] In certain embodiments, the antibodies described herein are prepared using cell-based immunoglobulins known in the art. As measured by a phosphorylation assay, for example, a cell-based phosphorylation assay described herein When activated, KIT activity, e.g., ligand-induced tyrosine phosphorylation of the KIT cytoplasmic domain, is inhibited by In certain embodiments, the antibodies described herein inhibit the Cell-based phosphorylation assays known in the art, such as the cell-based assays described herein, KIT activity, e.g., ligand activity of the KIT cytoplasmic domain, as measured by a phosphorylation assay of In certain embodiments, the compound inhibits agonist-induced tyrosine phosphorylation by about 35% to about 85% or 95%. Therefore, the antibodies or antigen-binding fragments thereof or conjugates thereof described herein are Cell-based phosphorylation assays known in the art, such as the cell-based assays described herein, less than about 600 pM, or less than about 500 pM, or less than about 250 pM as measured by a phosphorylation assay Less than 50% inhibitory concentration (IC 50 ) and KIT activity, e.g., ligand-induced thrombin production of the KIT cytoplasmic domain. In a specific embodiment, the IC 50 is approximately 550 pM or less than 200 pM In a specific embodiment, the IC 50 is in the range of about 50 pM to about 225 pM or 100 pM to about In a specific embodiment, the IC 50 is about 50 pM to about 550 pM, or about 50 The range is from about 150 pM to about 550 pM.

[0256] In a specific embodiment, the antibody or antigen-binding fragment thereof described herein or These conjugates (i) immunospecifically bind to a KIT polypeptide containing the D4 region of human KIT. (ii) inhibit KIT phosphorylation (e.g., tyrosine phosphorylation), and (iii) inhibit KIT linkage to KIT. In another specific embodiment, the antibody does not affect binding of the antibody to a target protein (e.g., SCF). Such antibodies do not inhibit KIT dimerization. Such antibodies may be expressed in CHO cells at an average titer of at least 0.5 μg / mL, e.g., at least 1.0 μg / mL. In a further specific embodiment, such antibodies can be recombinantly expressed by cells. and VH and VL domains that are non-immunogenic, e.g., The antibody does not contain T cell epitopes.

[0257] In other specific embodiments, an antibody or antigen-binding fragment thereof described herein or These conjugates immunospecifically bind to the monomeric form of KIT (e.g., human KIT). In certain embodiments, the antibodies described herein bind to a monomeric form of KIT (e.g., human KIT). In a specific embodiment, the antibodies or antibodies described herein do not immunospecifically bind to the antibody or antibody fragments described herein. The antigen-binding fragment or conjugate thereof may be a dimeric form of KIT (e.g., human KIT). In a specific embodiment, the antibodies or antibodies thereof described herein immunospecifically bind to The antigen-binding fragments of the present invention or conjugates thereof do not bind to KIT in its monomeric form but to KIT in its dimeric form. In one embodiment, the antibody specifically binds to KIT or a multimeric form of KIT. In one embodiment, the antibody has a higher affinity for KIT monomer than for KIT dimer. It has a higher affinity for KIT monomers than for multimers.

[0258] In a specific embodiment, the anti-KIT antibodies (or antigen-binding fragments thereof or or their conjugates) are native isoforms or native variants of KIT. The present invention relates to a method for producing a mammalian cell line (e.g., a mammalian cell line), which can be isolated from a mammal (e.g., a monkey, mouse, goat, donkey, dog, cat, rabbit, pig, rat, human, frog, or bird) In certain embodiments, the antibody specifically binds to a naturally occurring isoform or variant of the IT. The antibodies described herein immunospecifically bind to human KIT or a fragment thereof. In one embodiment, an anti-KIT antibody (or antigen-binding fragment thereof or a component thereof) described herein is The conjugate specifically binds to human KIT or a fragment thereof and binds to non-human KIT (e.g., monkey, mouse, etc.). goat, donkey, dog, cat, rabbit, pig, rat, or bird) or fragments thereof In a specific embodiment, the anti-KIT antibodies (or or an antigen-binding fragment thereof, or a conjugate thereof) specifically binds to human KIT or a fragment thereof. In one embodiment, the anti-KIT antibodies described herein specifically bind to mouse KIT. The antibodies include those directed against human KIT or fragments thereof (e.g., the D4 region of human KIT) and canine (dog) and non-human KIT. In one embodiment, the antibody specifically binds to primate (e.g., monkey) KIT. Anti-KIT antibodies are specific for human KIT or fragments thereof (e.g., the D4 region of human KIT) and canine (dog) KIT. In certain embodiments, the anti-KIT antibodies described herein bind to human KIT or its analogs. fragments of KIT (e.g., the D4 region of human KIT) and non-human primate (e.g., monkey) KIT. In certain embodiments, the anti-KIT antibodies described herein are directed against human KIT or a fragment thereof (e.g., specific for canine (dog) and non-human primate (e.g., monkey) KIT. It binds to mouse KIT or a fragment thereof (e.g., the D4 region of mouse KIT), but does not specifically bind to mouse KIT or a fragment thereof (e.g., the D4 region of mouse KIT). stomach.

[0259] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein may contain mutations, e.g. For example, somatic mutations associated with cancer (e.g., GIST), e.g., Ala and T at positions 502 and 503, The extracellular domain of human KIT contains a mutation in exon 9 of human KIT that duplicates the yr residue. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein bind to The extracellular domain of native human KIT and mutations, such as those associated with cancer (e.g., GIST), are described. Cellular mutations, e.g., exon 1 of human KIT, in which the Ala and Tyr residues at positions 502 and 503 are duplicated. It binds to the extracellular domain of human KIT containing a mutation in sequence 9 (e.g., Marcia et al. (2000) Am. J. Pathol. 156(3):791-795; and Debiec-Rychte See r et al. (2004) European Journal of Cancer. 40:689-695, which (both of which are incorporated herein by reference in their entireties).

[0260] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are glycosylated. In one embodiment, the antibody binds to the extracellular domain of human KIT, which is described herein. The antibody or antigen-binding fragment thereof described above is a human KIT antibody that binds to two different glycosylation sites in the extracellular domain of the human KIT. For example, different molecular weights of human IgGs that exhibit different glycosylation patterns can be bound to different forms of IgG. Two forms of human KIT have been observed by immunoblotting. Therefore, the antibodies described herein may have different glycosylation patterns, e.g., one form Specific for both of these forms of human KIT, one form being more glycosylated than the other. In certain embodiments, the antibodies or antigens thereof described herein can bind to the target protein. The binding fragment binds to the non-glycosylated extracellular domain of human KIT.

[0261] In certain embodiments, the antibodies described herein are those described in International Patent Application WO 2008 / 153926. Described KIT epitopes, e.g., amino acid sequences [ka] does not immunospecifically bind to an epitope consisting essentially of

[0262] In certain embodiments, the anti-KIT antibodies described herein include: SR-1 antibody (U.S. Patent Application Publication No. US 2003 / 0129998); 2007 / 0253951 A1; see International Patent Application Publication No. WO 2007 / 127317); Deutsche Sammlung vo n Mikroorganismen und Zellkulturen GmbH, DSM, Mascheroder Weg 1 b, D-38124 Braum Hybridoma cell lines DSM ACC 2007 and DSM ACC 2008 deposited in Schweig, Germany or anti-KIT antibodies obtained from DSM ACC 2009 (U.S. Pat. No. 5,545,533; International Patent Application Publication No. WO 99 / 09994). 2 / 021766); hybridoma cell line DSM ACC 2247 (or A3C6E2; Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSM, Mascheroder Weg 1 b, D-38124 Br The antibody produced by the antibody (U.S. Patent No. DSM ACC 2247) is See Patent No. 5,808,002); and the antibodies designated K27, K44, K45, K49, K57, K69, and K94. KIT antibodies (e.g., Blechman et al., Stem Cells, 1993, 11:12-21; Blechman et al., Cell, 1995, 80:103-113; Lev et al., Mol. Cell. Biol., 1993, 13:2224-2234; and (See State Patent Application Publication No. EP0548867 A2). In embodiments, the anti-KIT antibodies described herein comprise the CDRs of an antibody selected from such a group. In certain embodiments, the anti-KIT antibodies described herein do not include those from such groups. One or more (e.g., two, three, four, five, or six) CDRs (e.g., three V In another embodiment, the antibody described herein does not comprise any of the VL CDRs and / or the three VH CDRs. The binding activity of these antibodies is measured, for example, by competitive binding assays (e.g., ELISA). is not competitively inhibited by one of the In certain embodiments, the anti-KIT antibodies described herein are directed to the compounds disclosed in US Pat. No. 6,499,499, filed Dec. 22, 2010. It is not antibody Ab1 or Ab21 described in U.S. Provisional Application No. 61 / 426,387. In the present specification, the anti-KIT antibodies are described in: U.S. Provisional Application No. 61 / 426, filed December 22, 2010 Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, and A b12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, and Ab21, and March 25, 2011 Ab24 to Ab192 described in PCT International Patent Application No. PCT / US2011 / 29980 filed on the same day In certain embodiments, the anti-KIT antibody described herein is not an antibody selected from the group consisting of: are: Ab1, Ab2, and A, as described in U.S. Provisional Application No. 61 / 426,387, filed December 22, 2010 b3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17 , Ab18, Ab19, Ab20, and Ab21, and PCT international patent application PCT / CDRs of antibodies selected from the group consisting of Ab24 to Ab192 described in US2011 / 29980, or It does not contain one or more CDRs (e.g., three VL CDRs and / or three VH CDRs). In this regard, the anti-KIT antibodies described herein are disclosed in U.S. Provisional Application No. 6, filed December 22, 2010. No. 1 / 426,387 or PCT International Patent Application No. PCT / US2011 / 29980 filed March 25, 2011 the CDRs of an antibody selected from the antibodies (e.g., antibodies Ab1 to Ab21 and Ab24 to Ab192), or one or more CDRs (e.g., three VL CDRs and / or three VH CDRs), a VL chain region, or a VH chain region In certain embodiments, the anti-KIT antibodies described herein do not comprise the nucleotide sequence identified in the nucleotide sequence of the antibody fragment ... Antibodies Ab1 or Ab21, as described in U.S. Provisional Application No. 61 / 426,387, filed on comprises one, two, three, four, five, or six CDRs of antibody Ab1 or Ab21. In certain embodiments, the antibodies described herein are not antibodies comprising The antibodies 37M and 37C described in PCT International Patent Application No. PCT / US2012 / 022471 filed In certain embodiments, the antibodies described herein have a VL domain comprising SEQ ID NO: 32 or does not contain a VH domain comprising SEQ ID NO:31.

[0263] In certain embodiments, a KIT polypeptide (e.g., the D4 region of KIT, e.g., human KIT) The antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to Antibodies described in US 2008 / 0287309, such as antibodies 36C1, 84H7, 63C10, or 65A1 One or more (e.g., two, three, four, five, or six) CDRs (e.g., VL CDR1, VL CDR2, VL VH CDR1, VH CDR2, and VH CDR3).

[0264] In specific embodiments, the antibodies described herein are those described, for example, in U.S. Pat. No. 5,919,911 No. 5,489,516 or the American Type Culture Collection (ATC). CC) Human or human antibodies produced by hybridoma (BA7.3C.9) having accession number HB10716 In another specific embodiment, the antibodies described herein are, e.g., , the American Type described in U.S. Patent No. 5,919,911 or U.S. Patent No. 5,489,516 by a hybridoma (BA7.3C.9) with the American Type Culture Collection (ATCC) accession number HB10716. The CDRs of the antibody to be produced (e.g., VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or In another specific embodiment, the antibodies described herein comprise, for example, For example, U.S. Patent No. 5,919,911 or U.S. Patent No. 5,489,516 or U.S. Patent Application Publication No. US 2007 / 02 53951 A1 (e.g., ¶

[0032] or ¶

[0023] ) In further embodiments, the antibodies described herein are prepared in accordance with the methods described in, for example, U.S. Pat. American Type Culture, as described in U.S. Patent No. 5,919,911 or U.S. Patent No. 5,489,516 The hybridoma (BA7.3C.9) was produced by the American Type Culture Collection (ATCC) with accession number HB10716. It is not an antibody of an antibody.

[0265] In a specific embodiment, the antibodies described herein are those disclosed in U.S. Patent Application Publication No. US 2007 / 0253 In a specific embodiment, the antibody is not a humanized version of the SR-1 antibody described in US Pat. No. 951 A1. The antibodies described herein may be derived from the sequences referenced in U.S. Patent Application Publication No. US 2007 / 0253951 A1. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. In certain embodiments, the antibodies described herein do not comprise the amino acid sequence SEQ ID NOs: 2 and 4 or SEQ ID NOs: 2 and 4 mentioned in US Patent Application Publication No. US 2007 / 0253951 A1 and 6. In a specific embodiment, the antibodies described herein do not comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10, and at least 9 In certain embodiments, the present invention does not include one or more amino acid sequences that are 0.001% identical to the amino acid sequences described herein. The antibody may comprise one or more CDRs described in U.S. Patent Application Publication US 2007 / 0253951 A1, e.g., , amino acids 44 to 58 of SEQ ID NO:8 [ka] Amino acids 74 to 80 of SEQ ID NO: 8 [ka] Amino acids 111 to 121 of SEQ ID NO: 8 [ka] Amino acids 50 to 54 of SEQ ID NO: 10 [ka] Amino acids 69 to 85 of SEQ ID NO: 10 [ka] and / or amino acids 118 to 125 of SEQ ID NO: 10 [ka] wherein SEQ ID NOs: 8 and 10 are referenced in U.S. Patent Application Publication No. US 2007 / 0253951 A1. (See, for example,

[0032] or

[0023] .) Specific embodiments In the present specification, the antibodies are described in U.S. Patent Application Publication No. US 2007 / 0253951 A1. one or more CDRs, for example, amino acids 43 to 58 of SEQ ID NO: 2 (VL CDR1), amino acids 44 to 58 of SEQ ID NO: 2 (VL CDR2), amino acids 74 to 80 (VL CDR2), amino acids 113 to 121 (VL CDR3) of SEQ ID NO: 2, amino acids 50 to 52 (VL CDR4) of SEQ ID NO: 4 54 (VH CDR1), amino acids 69 to 85 (VH CDR2) of SEQ ID NO: 4, and / or amino acid 118 of SEQ ID NO: 4 125 (VH CDR3), wherein SEQ ID NOs: 2 and 4 are the same as those described in U.S. Patent Application Publication US 2007 / 025395 1 A1. In certain embodiments, the antibodies described herein are However, it is not an antibody of antibody SR-1 described in US Patent Application Publication No. US 2007 / 0253951 A1.

[0266] In a specific embodiment, the antibodies described herein are those disclosed in U.S. Pat. No. 6,989,248 or Anti-S100, ACK2, and ACK4 antibodies described in U.S. Pat. No. 7,449,309. In certain embodiments, the antibodies described herein are not antibodies of choice. In a specific embodiment, the antibody is not a human or humanized version of the antibody selected from the group. The antibodies described herein are those described in U.S. Patent No. 6,989,248 or U.S. Patent No. 7,449,309. one or more CDRs (e.g., three CDRs) of an antibody selected from the group consisting of: anti-S100, ACK2, and ACK4 antibodies The antibody is not an antibody comprising one VL CDR and / or three VH CDRs.

[0267] In some embodiments, competitive binding assays are used to help identify the target epitope of an antibody. or two antibodies, all numbers of different antibodies using either labeled antigen or labeled antibody. In a competitive binding assay, such as a competitive ELISA assay, which may be configured in a format Or, if an antibody recognizes sterically overlapping epitopes, it may be possible for another antibody, e.g., Antibodies that essentially bind to the same or overlapping epitopes as the antibody, e.g. For example, it can be determined whether the compound is competitively inhibited in a dose-dependent manner. Several types of competitive binding assays are known, for example: solid-phase direct or indirect radioimmunoassays. Immunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (Stahl (1983) Methods in Enzymology 9:242); solid-phase direct biotin-avidin EIA (K See Kirkland et al. (1986) J. Immunol. 137:3614; solid-phase direct labeling assay, solid-phase direct Labeled sandwich assay (Harlow and Lane, 1988; Antibodies: A Laboratory Manual) ies: A Laboratory Manual), Cold Spring Harbor Press); solid-state imaging using I-125 labeling solid-phase direct labeling RIA (see Morel et al. (1988) Mol. Immunol. 25(1):7); solid-phase direct biotin- Avidin EIA (Cheung et al. (1990) Virology 176:546); and direct label RIA (Moldenhauer et al. (1990) Scand J. Immunol. 32:77). Typically, such assays are A solid surface carrying either an unlabeled test immunoglobulin or a labeled reference immunoglobulin is used. Surface- or cell-bound purified antigen (e.g., KIT, e.g., the extracellular domain of KIT or D4 of KIT) Competitive inhibition involves the use of a region (a region) bound to a solid surface or cell in the presence of the test immunoglobulin. This can be measured by determining the amount of label that is bound to the test immunoglobulin. In some embodiments, when a competing antibody is present in excess, the antibody: Specific binding of the reference antibody to a common antigen is increased by at least 50-55%, 55-60%, 60-65%, In the general form of this assay, the antibody The antigen is immobilized on a 96-well plate. Then, a non-labeled antibody is added to prevent the labeled antibody from binding to the antigen. The ability of antibodies to bind to the antibody is measured using radioactive or enzyme labels. See, e.g., Wagener et al., J. Immunol., 1983, 130:2308-2315; Wagener et al., J. I Munol. Methods, 1984, 68:269-274; Kuroki et al., Cancer Res., 1990, 50:4872-48 79; Kuroki et al., Immunol. Invest., 1992, 21:523-538; Kuroki et al., Hybridom a, 1992, 11:391-407, and Using Antibodies: A Laboratory Manual. y Manual), Ed Harlow and David Lane, eds. (Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY, 1999), pp. 386-389.

[0268] In some embodiments, the D4 region of a KIT polypeptide (e.g., a human KIT polypeptide) is immunospecifically The antibodies described herein that bind heterologously to the VL chain region (e.g., any of SEQ ID NOS: 7 to 10) may be or one of the VH chain regions (for example, any one of SEQ ID NOs: 2 to 6), or a combination of the three thereof. The VL CDR or its three VH CDRs can be described in full by reference. See Rader et al., 1998, Proc. Natl. Acad. Sci. USA, 9, which is incorporated herein in its entirety. 5: 8910-8915, which refers to the complementary light or heavy chains as human light chains, respectively. or heavy chain library and have an affinity as high as or higher than that of the original antibody. Humanization of a murine anti-αvβ3 antibody by generating high-affinity humanized antibody variants Clackson et al., 1999, incorporated herein by reference in its entirety. See also, 91, Nature, 352:624-628, which describes the use of specific VL (or VH) domains. By using the antibody to screen a library of complementary variable domains, specific antibodies can be identified. From this screening, specific VH domains can be identified. 14 new partners of the main and 13 new partners of the specific VL domain were obtained, These were strong binders as judged by ELISA.

[0269] Thus, in some embodiments, provided herein are KIT polypeptides (e.g., an antibody that immunospecifically binds to the D4 region of human KIT polypeptide, the antibody being selected from SEQ ID NO: 7 or 8 In some embodiments, the antibody of the present invention comprises a VL domain comprising the amino acid sequence of Provided herein are antibodies directed to the D4 region of a KIT polypeptide (e.g., a human KIT polypeptide). An antibody that specifically binds to a VH domain comprising the amino acid sequence of SEQ ID NO: 4 or 5. It is an antibody.

[0270] In some embodiments, the CDRs of the antibodies described herein are selected from the group consisting of the CDRs of Chothia and Lesk (1987, J. Mol. Biol., 196:901-917), which is referred to herein as "Chothi The VH chain and VL chain regions are referred to as "a CDR" (see, e.g., U.S. Patent No. 7,709,226). Using the Kabat numbering system for numbering amino acid residues in a region, Chothia C in an antibody heavy chain molecule The DRs typically consist of amino acid positions 26-32 ("CDR1"), amino acid positions 53-55 ("CDR2"), and amino acid positions 60-65 ("CDR3"). The amino acid residues in the VH and VL chain regions are numbered as follows: Using the Kabat numbering system, the Chothia CDR within an antibody light chain molecule is typically located at amino acid position 26. 33 (CDR1), amino acid positions 50-52 (CDR2), and amino acid positions 91-96 (CDR3).

[0271] In a specific embodiment, the CDRs along the VH and / or VL regions of the antibodies described herein The position is such that immunospecific binding to KIT (e.g., the D4 region of human KIT) is maintained (e.g., Substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80% , at least 90%, at least 95% (maintained) one, two, three, or four amino acids. For example, in one embodiment, the CDRs of the antibodies described herein may differ by The position to be determined is such that immunospecific binding to KIT (e.g., D4 region) is maintained (e.g., substantially Typically, for example, at least 50%, at least 60%, at least 70%, at least 80%, moving the N-terminal and / or C-terminal boundaries of the CDRs, as long as the N-terminal and / or C-terminal boundaries are maintained at least 90%, and at least 95% By changing the CDR positions shown in Figures 3A-3I, only 1, 2, 3, or 4 amino acids are added. may vary.

[0272] In specific aspects, provided herein are antibody light and heavy chains, e.g., separate In a specific embodiment, the light chain is an antibody comprising a light chain and a heavy chain. In another specific embodiment, the light chain of the antibody described herein is a kappa light chain. The light chain of the antibody described herein is a lambda light chain. The light chain of the antibody described above is a human kappa light chain or a human lambda light chain. and a KIT polypeptide (e.g., KIT, e.g., the D4 region of human KIT (e.g., SEQ ID NO: 15) The antibodies described herein that immunospecifically bind to a KIT polypeptide comprising a light chain include Here, the amino acid sequence of the VL chain region may be any amino acid sequence described herein (e.g., the sequence Nos. 7, 8, 9, or 10), wherein the constant region of the light chain comprises the amino acid sequence of the human kappa light chain constant region. Non-limiting examples of human light chain constant region sequences are described in the art, e.g., See, e.g., U.S. Pat. No. 5,693,780 and Kabat et al. (1991), Proteins of Immunological Interest Sequences of Proteins of Immunological Interest, 5th Edition, US Department of of Health and Human Services, NIH Publication No. 91-3242.

[0273] With respect to the heavy chain, in a specific embodiment, the heavy chain of the antibodies described herein is alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chains. In another specific embodiment, the heavy chain of the described antibody is human alpha (α), It can include delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chains. In certain embodiments, a KIT polypeptide (e.g., a KIT, e.g., a D4 region of human KIT (e.g., and immunospecifically binds to a KIT polypeptide, including a KIT polypeptide comprising SEQ ID NO: 15). The antibodies described herein comprise a heavy chain, wherein the amino acid sequence of the VH chain region is The heavy chain may comprise any of the amino acid sequences described herein (for example, any of SEQ ID NOS: 2 to 6), The constant region of the human heavy chain comprises the amino acid sequence of the human gamma (γ) heavy chain constant region. Non-limiting examples of arrays are described in the art, for example, U.S. Pat. No. 5,693,780 and and Kabat et al. (1991), Sequences of Proteins of Immunological Interest of Immunological Interest), 5th ed., US Department of Health and Human Services, See NIH Publication No. 91-3242.

[0274] In a specific embodiment, a KIT polypeptide (e.g., the D4 region of KIT, e.g., human KIT) is The antibodies described herein that immunospecifically bind to any of the amino acid sequences described herein. The constant region comprises a VL chain region and a VH chain region, each of which contains a sequence, and the constant region is selected from the group consisting of IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, or human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulins In another specific embodiment, the KI comprises the amino acid sequence of the constant region of an immunoglobulin molecule. The present invention provides a method for immunospecifically binding to a T polypeptide (e.g., the D4 region of KIT, e.g., human KIT). The antibodies described herein comprise a VL chain region and a VH chain region comprising any of the amino acid sequences described herein. The constant region is a region that encodes an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, an immunoglobulin any class of globulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or It includes the amino acid sequence of the constant region of any subclass (e.g., IgG2a and IgG2b). In embodiments, the constant region is selected from the group consisting of human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulins. immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or the amino acid sequence of the constant region of any subclass (e.g., IgG2a and IgG2b) Includes:

[0275] In yet another specific embodiment, a KIT polypeptide (e.g., KIT, e.g., human KIT) is The antibodies described herein that immunospecifically bind to the D4 region of any of the antibodies described herein. amino acid sequence (e.g., any one of SEQ ID NOs: 2 to 6 and / or any one of SEQ ID NOs: 7 to 10) wherein the constant region comprises a VL chain region and a VH chain region comprising the constant region of human IgG1 or human IgG4. In certain embodiments, the amino acid sequence of a KIT polypeptide (e.g., KIT, For example, the antibodies described herein that immunospecifically bind to the D4 region of human KIT are and VL chain regions and VH chain regions comprising any of the amino acid sequences described herein, wherein the constant region is a human It contains the amino acid sequence of the constant region of human IgG1.

[0276] In a specific embodiment, a KIT polypeptide, e.g., a human KIT polypeptide, e.g., , as described herein, which immunospecifically binds to the D4 region of KIT (e.g., human KIT, e.g., SEQ ID NO: 15). The antibodies described herein have human framework regions or are derived from human framework regions. framework regions (e.g., framework regions of the VL domain and / or VH domain) Non-limiting examples of human framework regions are described in the art, e.g., For example, Kabat et al. (1991), Sequences of Proteins of Immunological Interest, s of Immunological Interest), 5th ed., US Department of Health and Human Services See, e.g., NIH Publication No. 91-3242. In some embodiments, The antibodies described herein may be primate (e.g., non-human primate) framework regions or may be primate (e.g., framework regions (e.g., VL domains) derived from non-human primate framework regions The VH domains of the nucleotide sequence are also included in the nucleotide sequence.

[0277] In some examples, the antibodies described herein are administered to a primate (e.g., a non-human primate, e.g., Apes (e.g., Old World apes) may be framework regions, but primates (e.g., non-human apes) may also be framework regions. framework regions (e.g., VL domains) that are not derived from human primate framework regions. The VH domains of the ribonucleotides contain the VH domain framework regions.

[0278] In certain instances, for any of these antibodies described herein, the VL chain region comprises a non- Also include human primate (e.g., ape, e.g., Old World ape) framework regions. Derived from non-human primate (e.g., ape, e.g., Old World ape) framework regions In certain other embodiments, the VH chain region is derived from a non-human primate (e.g., ape, e.g., framework regions from non-human primates (e.g., apes, For example, Old World apes) and do not originate from the framework region.

[0279] Non-limiting examples of non-human primate framework regions include those from Old World apes, e.g., Chironomids. Pan troglodytes, bonobo (Pan paniscus), or western gorilla (Gorilla gorilla) a); chimpanzees; Old World monkeys, such as Old World monkeys of the genus Macaca; and Macaca fascicularis Examples include those derived from Macaca cynomolgus. Non-limiting examples of framework sequences are described in U.S. Patent Application Publication No. US 2005 / 0208625. do.

[0280] In some embodiments, also provided herein are KIT polypeptides (e.g., KIT, an antibody that immunospecifically binds to a VL chain region or a D4 region of human KIT, for example, An antibody that contains one or more amino acid residue substitutions in the VH chain region, for example, in the CDR or FR. In certain embodiments, there are no amino acid residue substitutions within the CDRs. In one embodiment, the amino acid substitutions are all in the FR (see, e.g., Tables 5A-6B). The amino acid substitutions are conservative amino acid substitutions.

[0281] As used herein, a "conservative amino acid substitution" refers to a substitution in which the amino acid residue is replaced by a similar An amino acid substitution is one in which an amino acid residue has been substituted with an amino acid residue having a charged side chain. Families of amino acid residues having charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, hyaluronic acid, etc.). amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), uncharged Amino acids with polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, Contains amino acids (amino acids, histidine).

[0282] In certain embodiments, the glycosylation of the antibodies described herein is modified. For example, An aglycoslated antibody can be made (i.e., the antibody lacks glycosylation) or or at one or more glycosylation sites that abolishes glycosylation at one or more glycosylation sites Antibodies can be generated that contain mutations or substitutions of: For example, the affinity of an antibody for a target antigen (e.g., human KIT, e.g., the D4 region of human KIT) can be determined by Such carbohydrate modifications can be, for example, by modifying one or more groups within the antibody sequence. This can be achieved by modifying glycosylation sites, for example by modifying one or more variable regions. Loss of glycosylation sites in regions (e.g., VL and / or VH CDRs or VL and / or VH FRs) and making one or more amino acid substitutions resulting in abolishing glycosylation at that site. Such aglycosylation can be achieved by deglycosylating the antigen (e.g., human KIT, e.g., human This method can increase the affinity of antibodies to the target protein (the D4 region of KIT). Further details are described in US Patent Nos. 5,714,350 and 6,350,861.

[0283] Glycosylation can be N-linked (or asparagine-linked) glycosylation or O-linked glycosylation. N-linked glycosylation can occur by asparagine amino acid substitution in polypeptides. O-linked glycosylation involves carbohydrate modification at the side chain NH2 group of the acid. or with carbohydrate modification at the hydroxyl group on the side chain of the hydroxylysine amino acid.

[0284] In specific embodiments, the VH regions of the antibodies described herein (e.g., SEQ ID NOs: 2, 3, 4) , 5, or 6) or an asparagine (N) in the VL region (e.g., SEQ ID NO: 7, 8, 9, or 10). ) residue can be replaced with serine (S) or another amino acid (e.g., alanine, glycine, glutamine, threonine, In another specific embodiment, the amino acid sequence of the present invention is substituted with a cysteine, tyrosine, or cysteine. VH CDRs of the antibody described in (for example, VH CDR1, VH CDR2, VH CDR3, VH CDR4, VH CDR5, VH CDR6, VH CDR7, VH CDR8, VH CDR9, VH CDR10, VH CDR11, VH CDR12, VH CDR13, VH CDR14, VH CDR15, VH CDR16, VH CDR17, VH C DR2, and / or VH CDR3), and / or VL CDR (e.g., SEQ ID NOs: 19 to 21, respectively). Asparagine (N) residues in VL CDR1, VL CDR2, and / or VL CDR3, including the sequences phosphorus (S) or another amino acid (e.g., alanine, glycine, glutamine, threonine, thyroxine) In another specific embodiment, the antibody described herein is substituted with a cysteine ​​(e.g., cysteine). VH FRs (e.g., VH FR1, VH FR2, VH FR3 and / or VH FR4 shown in Tables 5A, 5C, and 6B) of FR4) and / or VL FR (e.g., VL FR1, VL FR2, VL FR3, VL FR4, VL FR5, VL FR6, VL FR7, VL FR8, VL FR9, VL FR10, VL FR11, VL FR12, VL FR13, VL FR14, VL FR15, VL FR16, VL FR17, and / or VL FR4) to a serine (S) or another amino acid (e.g. , alanine, glycine, glutamine, threonine, tyrosine, cysteine).

[0285] In one embodiment, the aglycosylated antibody is expressed in a bacterial cell that lacks the necessary glycosylation machinery. Cells with modified glycosylation machinery are known in the art. and the cells are used as host cells for expressing the recombinant antibodies described herein. Alternatively, antibodies can be produced using modified glycosylation sequences, thereby producing antibodies with altered glycosylation. See, for example, Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, and European Patent: EP 1,176,195; PCT Publication WO 03 / 0358 35; WO 99 / 54342.

[0286] In certain embodiments, one or more modifications are made to the Fc region of an antibody described herein, Typically, the antibody has one or more functional properties, such as serum half-life, complement fixation, Fc receptor binding, and / or These modifications can alter antibody-dependent cellular cytotoxicity. This is described in published application WO 2008 / 153926 A2.

[0287] In a specific embodiment, the constant region and / or light region of the heavy chain of an antibody described herein Asparagine (N) residues in the constant region of , glycine, glutamine, threonine, tyrosine, cysteine).

[0288] In a specific embodiment, aspartate amino acids within the heavy and / or light chain regions of the antibodies described herein are The amino acid residue (N) can be replaced with serine (S) or another amino acid (e.g., alanine, glycine, glutamine, etc.). threonine, tyrosine, cysteine).

[0289] Provided herein are antibodies capable of immunospecifically binding to the KIT antigen and modulating KIT activity. In certain embodiments, the antibodies provided herein are antibodies capable of binding to the KIT polypeptide. The antibody binds immunospecifically to a human KIT polypeptide, for example, and inhibits KIT activity. The activity may be any activity of KIT known or described in the art, such as KIT receptor dimerization, KIT Receptor phosphorylation (tyrosine phosphorylation), downstream signaling of the KIT receptor (e.g., AKT, MAP) K / ERK, Ras, Stat1, Stat3, or Stat5 signaling), KIT ligand (e.g., SCF) induction Inducible transcriptional regulation (e.g., SCF-induced transcriptional activation of c-Myc), induction or enhancement of cell proliferation, or KIT activity or KIT function are used interchangeably herein. In embodiments, KIT activity is induced by KIT ligand (e.g., SCF) binding to the KIT receptor. In certain embodiments, KIT activity is induced, e.g., by dimerization and constitutively active KIT. It can be induced or enhanced by gain-of-function mutations that can result in IT signaling (e.g., See, for example, Mol et al., J. Biol. Chem., 2003, 278:31461-31464; Hirota et al., J. Patho (See, e.g., logy, 2001, 193:505-510). Such gain of function results in the KIT receptor dimerization. Somatization and KIT signaling occur in the absence of KIT ligand (e.g., SCF) binding to the KIT receptor. In some embodiments, inhibition of KIT activity or signaling may be possible. The increase is due to the high expression (or overexpression) of KIT receptor, which leads to the binding of KIT ligands (e.g., For example, this can occur in the absence of SCF binding. than the expression level of reference cells known to have normal KIT expression or KIT activity, or a population of cells or samples known to have normal KIT expression or KIT activity. at least about 10%, 20%, 30%, 40%, 50%, 60% higher than the average expression level of KIT in , 70%, 80%, 90%, or 100% greater expression levels. or methods known to those skilled in the art (e.g., Western blotting or immunohistochemistry) In certain embodiments, KIT activity can be assessed by: The activity can result in cell transformation, neoplasia, and tumorigenesis. In some embodiments, the higher than normal KIT activity results in other KIT-related disorders or diseases. It is possible.

[0290] In certain embodiments, the anti-KIT antibodies described herein bind to KIT ligands to the KIT receptor. In some embodiments, the compounds described herein do not block or inhibit SCF binding. Anti-KIT antibodies inhibit KIT ligand (e.g., SCF) binding to the KIT receptor to a minimal extent (e.g., In certain embodiments, the method described herein inhibits or reduces the level of IL-1 by only about 2% or less than about 3%. The anti-KIT antibody described in 1. induces or enhances dissociation of a KIT ligand (e.g., SCF) from the KIT receptor. In certain embodiments, the anti-KIT antibodies described herein inhibit KIT release from the KIT receptor. Induce or enhance only slightly (e.g., less than about 2% or 3%) the dissociation of a target protein (e.g., SCF). do not.

[0291] In certain embodiments, the anti-KIT antibodies described herein block or inhibit KIT receptor dimerization. In certain embodiments, the anti-KIT antibodies described herein do not inhibit KIT receptor dimerization. only slightly (e.g., less than about 2% or 3% or within the standard error or deviation) inhibiting In certain embodiments, the anti-KIT antibodies described herein do not inhibit or reduce KIT receptor activity. In certain embodiments, the anti-KIT antibodies described herein do not induce or enhance dimer dissociation. The body may induce negligible (e.g., less than about 2% or 3% or less than standard error) dissociation of the KIT receptor dimer. In certain embodiments, the agonist induces or enhances only a specific gene expression level (or within a deviation thereof). The anti-KIT antibody can specifically bind to the KIT receptor dimer and inhibit KIT receptor dimerization. In certain embodiments, the anti-KIT antibodies described herein do not block or inhibit KIT receptor activity. It can specifically bind to the dimer of the KIT receptor and does not block or inhibit KIT receptor dimerization.

[0292] In some embodiments, as inhibitors of KIT activity, the antibodies described herein inhibit KIT dimerization. Typically, the KIT receptor dimer is capable of blocking or inhibiting (e.g., partially inhibiting) The activation is induced when a KIT ligand binds to KIT. In the present invention, the antibodies described herein specifically bind to KIT and are capable of binding to KIT, as described herein or by those skilled in the art. The presence of KIT ligand stimulation was assessed by well-known methods, for example, immunoprecipitation assays. In the presence of KIT, without antibody or with irrelevant antibody (e.g., antibody that does not immunospecifically bind to KIT), and inhibiting KIT receptor dimerization by at least about 5%, 10%, 15%, or 20% relative to KIT receptor dimerization. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95%, 98%, or 99% blocking or inhibiting (e.g., partially inhibiting). In certain embodiments, the antibodies described herein specifically bind to KIT and inhibit the dimerization of the KIT receptor. The antibodies described herein partially inhibit the dimerization of KIT receptors by about 25% to 75%. Blockade or inhibition (e.g., partial inhibition) of activation can be assessed in the presence of KIT ligand stimulation. For example, cells expressing KIT can be cultured in the presence or absence of an anti-KIT antibody described herein. In one embodiment, the antibody is contacted with a KIT ligand in the presence of the antibody, and the level of KIT receptor dimerization is determined. In the present specification, KIT ligand-induced KIT receptor dimerization in the absence of anti-KIT antibodies is or known to those skilled in the art (e.g., immunoprecipitation assays). at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, or greater than KIT receptor dimerization in the presence of a KIT antibody; 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more abundant. Tyrosine phosphorylation of one or more residues in the KIT receptor can be an indicator of KIT receptor dimerization.

[0293] In certain embodiments, the antibodies described herein can be administered to patients as described herein and / or by those skilled in the art. When assessed by known methods, the effect of KIT ligand stimulation, without antibody or irrelevant KIT activity compared to KIT activity in the presence of an antibody (e.g., an antibody that does not immunospecifically bind to KIT). At least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% inhibition (e.g., partial inhibition) In certain embodiments, the antibodies described herein can inhibit the In the presence of KIT ligand stimulation, as assessed by methods described and / or known to those skilled in the art, KIT activity in the absence of antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to KIT) inhibits (e.g., partially inhibits) KIT activity by at least about 25% to about 65% compared to Non-limiting examples of KIT activity include KIT receptor phosphorylation, KIT receptor signaling, and the like. Transduction, KIT ligand (e.g., SCF)-mediated cell proliferation, KIT ligand (e.g., SCF)-mediated cell These include survival, and transcriptional activation of KIT target genes (e.g., c-Myc).

[0294] As an inhibitor of KIT activity, the antibody (or antigen-binding fragment thereof, or conjugates thereof) inhibit the phosphorylation of KIT, specifically, one or more kinases in the cytoplasmic domain of KIT. Blocking (e.g., partially blocking) or inhibiting (e.g., inhibiting) tyrosine phosphorylation of residues Dimerization and phosphorylation of the KIT receptor are usually mediated by KIT ligation. However, in some embodiments, the KIT receptor is induced when the KIT receptor binds to the Dimerization and / or phosphorylation can occur independently of KIT ligand binding to the KIT receptor. For example, KIT receptor dimerization and / or phosphorylation may be mediated by KIT gain-of-function mutations or It may be caused by overexpression.

[0295] For example, a chimeric protein in the cytoplasmic domain of mouse KIT that can be phosphorylated upon ligand stimulation. Non-limiting examples of lysine residues include 544, 546, 552, 567, 569, 577, 608, 645, 671, 6 74, 702, 719, 728, 745, 772, 821, 844, 853, 868, 878, 898, and 934 (U (See Eda et al., Blood, 2002, 99:3342-3349). The corresponding tyrosine residues in can be readily determined, e.g., by ligand stimulation. The cytoplasmic domain of human KIT (e.g., GenBank accession number P10721) can be phosphorylated. Non-limiting examples of tyrosine residues in the amino acid sequence include residues 568, 570, 703, 721, 730, 747, 823, and the like. , 900, and 936. In specific embodiments, the anti-KIT antibodies described herein can inhibit receptor phosphorylation at tyrosine residue 719 of mouse KIT. In a specific embodiment, the anti-KIT antibodies described herein target tyrosine residue 703 of human KIT. or inhibiting receptor phosphorylation at 721.

[0296] Thus, in a specific embodiment, the antibodies (or antigen-binding fragments thereof) described herein are The antibody or antibody fragment thereof specifically binds to KIT and is capable of binding to the antibody or antibody fragments described herein. The antibody can be isolated by methods known to those skilled in the art, such as the ELISA assay described in Section 6, or immunoblotting. As assessed by a stimulating assay, the presence of KIT ligand stimulation, without antibody, or irrelevant phosphorylation in the presence of a specific antibody (e.g., an antibody that does not immunospecifically bind to KIT) Tyrosine phosphorylation in the cytoplasmic domain is at least about 10%, 15%, 20%, 25%, 30% ,35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%, In certain embodiments, the antibodies described herein block or inhibit 98% or 99% of the target protein. specifically binds to KIT and is capable of binding to a target protein, such as a protein that binds to a target protein, and that binds ... When assessed by ELISA assay or immunoblotting assay, KIT tyrosine phosphorylation in the cytoplasmic domain of at least about 25%, optionally about 65% or 75% In certain embodiments, the antibodies described herein are specific for KIT. and selectively bind to the target protein by methods described herein or known to those of skill in the art, e.g., methods described in Section 6. The cytoplasmic expression of KIT was assessed by ELISA assay or immunoblotting assay. The tyrosine phosphorylation of the domain is blocked or inhibited by at least about 25% to about 80%. In embodiments, the antibodies described herein specifically bind to KIT and are Methods (e.g., the recombinant human ... The cytoplasmic domain of KIT is expressed as a cytoplasmic domain of KIT, as assessed by a phosphorylation inhibition assay (phosphorylation inhibition assay) or by methods known to those skilled in the art. tyrosine phosphorylation of ATP at less than about 600 pM, or less than about 550 pM, or less than about 500 pM, or less than about 400 pM IC less than pM or less than about 300 pM 50 In a specific embodiment, the present invention The antibodies described herein specifically bind to KIT and inhibit tyrosine phosphorylation of the cytoplasmic domain of KIT. , IC<approximately 600 pM 50 In a specific embodiment, the method described herein is The antibody specifically binds to KIT and inhibits tyrosine phosphorylation of the cytoplasmic domain of KIT at approximately 550 pM. Less than IC 50 In a specific embodiment, the antibodies described herein block or inhibit It specifically binds to KIT and inhibits tyrosine phosphorylation of the cytoplasmic domain of KIT at a concentration of about 100 pM to about 500 pM. IC in the range of about 25 pM to about 550 pM, or about 40 pM to about 600 pM, or about 50 pM to about 350 pM 50 or For example, IC for inhibition of tyrosine phosphorylation 50 recombinantly expresses KIT Lysates from cells, e.g., CHO cells, can be prepared, for example, using the Tyrosine kinase inhibitors described in Section 6 below. This can be determined by assaying with an ELISA that detects phosphorylation of ATP. In an embodiment, cells recombinantly expressing KIT, e.g., CHO cells, are selected, e.g., To select for cells with high KIT expression, we used 100% ATP-dependent phosphoproteins (PtPs) in the phospho-inhibition assay. In some embodiments, the cells are selected for use in a phosphorylation inhibition assay. They are not screened beforehand.

[0297] In a specific embodiment, an antibody (or antigen-binding fragment thereof) described herein is These conjugates) specifically bind to KIT and are suitable for use in the methods described herein or known to those skilled in the art. methods such as the ELISA assay described in Section 6, or immunoblotting assays. When assessed by (i), in the presence of KIT ligand stimulation, no antibody or an irrelevant antibody (e.g. cytoplasmic domain of KIT compared to phosphorylation in the presence of an antibody that does not immunospecifically bind to KIT. tyrosine phosphorylation of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% reduction In a specific embodiment, the antibodies (or antigen-binding fragments thereof) described herein or conjugates thereof) specifically bind to KIT and are described herein or by those skilled in the art. ELISA assays, as described in Section 6, or immunoblotting. As assessed by the assay, in the presence of KIT ligand stimulation, no antibody or an irrelevant antibody ( For example, the cytoplasmic domain of KIT is phosphorylated in the presence of an antibody that does not immunospecifically bind to KIT. Reduce main tyrosine phosphorylation by at least about 25% or 35%, optionally by about 75% .

[0298] In a specific embodiment, the antibodies described herein specifically bind to KIT and and assessing the activity of the antibody by methods described in the literature or known to those skilled in the art, for example, by immunoblotting assay. When the KIT ligand stimulation was detected, no antibody or an irrelevant antibody (e.g., an antibody specific for KIT) was detected. The phosphorylation of one or more of the cytoplasmic domains of KIT was observed compared to that observed in the presence of a non-specifically binding antibody. The phosphorylation of lysine residues is suppressed 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% blocking In a specific embodiment, the cytoplasmic expression of KIT by the antibodies described herein is inhibited. Blocking or inhibiting (e.g., partially inhibiting) phosphorylation of one or more tyrosine residues in the domain is achieved by KIT ligation. For example, KIT-expressing cells can be evaluated by the method described herein. contacting the KIT ligand with or without the anti-KIT antibody described above, and In one embodiment, the level of phosphorylation of one or more tyrosine residues in the In this case, the method described herein or known to those skilled in the art (e.g., immunoblotting assay) can be used. When assessed by (a), in the presence of KIT ligand stimulation, no antibody or an irrelevant antibody (e.g., Phosphorylation in the absence of anti-KIT antibodies compared with the presence of antibodies that do not immunospecifically bind to KIT. KIT ligand-induced phosphorylation of one or more tyrosine residues in the cytoplasmic domain of KIT under KIT ligand-induced ligation of one or more tyrosine residues in the cytoplasmic domain of KIT in the presence of IT antibodies At least about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, or 3.5 times higher than phosphorylation , 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x , 70, 80, 90, or 100 times more.

[0299] In a specific embodiment, an antibody (or antigen-binding fragment thereof) described herein is These conjugates) specifically bind to KIT and are suitable for use in the methods described herein or known to those skilled in the art. When assessed by the method of ) to enhance KIT receptor internalization by at least about 5%, 10%, 15%, 20%, or 30% compared to internalization in the presence of %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% In specific embodiments, the gene expression level of the present invention is induced or enhanced by 90%, 95%, 98%, or 99%. The antibody (or antigen-binding fragment thereof, or conjugate thereof) described in specifically bind to unrelated proteins as assessed by methods described herein or known to those of skill in the art. KIT receptors compared to internalization in the presence of antibodies (e.g., antibodies that do not immunospecifically bind to KIT). Induce or enhance internalization by at least about 25% or 35%, optionally up to about 75%. In embodiments, the antibodies described herein specifically bind to KIT and are or irrelevant antibodies (e.g., antibodies immunospecific for KIT) as assessed by methods known to those skilled in the art. Inhibiting KIT receptor internalization by at least about 1-fold compared to internalization in the presence of an antibody that does not bind to the KIT receptor. , 1.2x, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x , 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x Techniques for quantifying or visualizing cell surface receptors are well known in the art. These include a variety of fluorescent and radioactive techniques. For example, one method involves radiolabeling cells. Alternatively, the natural ligand of the receptor may be incubated with an anti-receptor antibody. can be conjugated to a fluorescent molecule or a radioactive label and incubated with cells. Additional receptor internalization assays are known in the art, see, for example, Jimenez et al. Biochemical Pharmacology, 1999, 57:1125-1131; Bernhagen et al., Nature Medicine, 2007, 13:587-596; and Conway et al., J. Cell Physiol., 2001, 189:341-5 5.

[0300] In a specific embodiment, the antibodies described herein specifically bind to KIT and The results were evaluated by methods described in the literature or known to those skilled in the art (e.g., pulse-chase assays). turnover in the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to KIT) Increasing KIT receptor turnover by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% In a specific embodiment, the antibodies described herein (if The antibody (or an antigen-binding fragment thereof, or a conjugate thereof) specifically binds to KIT and is The results were evaluated by methods described in the literature or known to those skilled in the art (e.g., pulse-chase assays). turnover in the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to KIT) Induce or induce KIT receptor turnover by at least about 25% or 35%, optionally by about 75%, compared to In a specific embodiment, the antibodies described herein specifically bind to KIT. and by methods described herein or known to those skilled in the art (e.g., pulse-chase assays). in the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to KIT) when assessed by and increasing KIT receptor turnover by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, or 1-fold compared to the turnover of .5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 2 Induce or enhance 0-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. Methods for determining metabolic turnover are well known in the art. For example, The cells 35S-EXPRESS Protein Labeling Mix (NEG772, NEN Life Science Products) The cells can be pulse-labeled with IgG, washed, and chased with unlabeled medium for a period of time, after which Protein lysates from labeled cells were immunoprecipitated with anti-KIT antibody and separated by SDS-PAGE. , visualized (e.g., by exposure to a PhosphoImager screen (Molecular Dynamics) Scanning was performed using a Typhoon 8600 scanner (Amersham) and imaged using ImageQuant software (Molecule lar Dynamics) (see, e.g., Chan et al., Development, 2004, 131:5551 -5560).

[0301] In a specific embodiment, the antibodies described herein specifically bind to KIT and The results were evaluated by methods described in the literature or known to those skilled in the art (e.g., pulse-chase assays). When compared with degradation in the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to KIT), In total, the KIT receptor degradation is inhibited 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% In a specific embodiment, the antibodies described herein induce or enhance KIT-specific and selectively bind to the target protein using methods described herein or known to those skilled in the art (e.g., pulse-chase assay). When assessed by (a), the presence of irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to KIT) inhibits KIT receptor degradation by at least about 25% or 35%, optionally about 75%, compared to degradation in the presence of In a specific embodiment, the antibodies described herein induce or enhance KIT-specific and then reacting with the antibody by methods described herein or known to those skilled in the art (e.g., pulse-chase assay). irrelevant antibodies (e.g., antibodies that do not immunospecifically bind to KIT) as assessed by ELISA. inhibits KIT receptor degradation by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, or , 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x , 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold induction or enhancement. quantitating or quantifying the ubiquitination and / or degradation (e.g., the kinetics or rate of degradation) of cell surface receptors; Techniques for monitoring are well known in the art and include various fluorescent and radioactive techniques. (See, for example, International Patent Application Publication No. WO 2008 / 153926 A2). Face experiment or 125 Experiments using radiolabeled ligands such as I-SCF were performed to detect the degradation of KIT. can be quantitatively measured.

[0302] Furthermore, signaling events downstream of KIT receptor phosphorylation may serve as indicators of KIT activity. For example, KIT ligand (e.g., SCF) binding to its receptor KIT. For example, Src family kinases, phosphatidylinositol (PI) 3-kinases, and Several different kinases, including members of the Ras and Ras mitogen-activated protein kinases (MAPKs), (Munugalavadla et al., Mol. Cell. Biol., 2005, 25:6 (See also 747-6759.) Phosphotyrosines in the cytoplasmic domain of KIT are involved in SH2 domain This SH2 domain-containing protein can provide a binding site for , a protein in the p21Ras mitogen-activated protein kinase (MAPK) pathway, and PI 3-kinase p85 subunit of phospholipase C-gamma 1, Grb2 adaptor protein, Src phagocytosis These include, but are not limited to, SFK, Cbl, CRKL, p62Dok-1, SHP1, and SHP2. It is not defined (see Ueda et al., Blood, 2002, 99:3342-3349).

[0303] Thus, in some embodiments, the anti-KIT antibodies described herein act as inhibitors of KIT activity. IT antibodies inhibit the signaling of members of the Src family kinases, PI 3-kinase, or Ras-MAPK. In certain embodiments, the IL-11 receptor agonist acts as an inhibitor of KIT activity. The anti-KIT antibodies described herein are directed against the cytoplasmic domain of KIT and contain one or more SH2 domains. Proteins involved, such as proteins in the p21Ras-MAPK pathway, the p85 subunit of PI 3-kinase, phospholipase C-gamma 1, Grb2 adaptor protein, SFK members, Cbl, CRKL , p62Dok-1, SHP1, and SHP2 can be inhibited from binding (or interacting). In certain embodiments, the anti-KIT antibodies described herein that act as inhibitors of KIT activity include: one or more SH2 domain-containing proteins by KIT, e.g., proteins of the p21Ras-MAPK pathway; p85 subunit of PI 3-kinase, phospholipase C-gamma 1, Grb2 adaptor protein It can inhibit the activation of proteins, SFK members, Cbl, CRKL, p62Dok-1, SHP1, and SHP2. Cut.

[0304] In certain embodiments, the anti-KIT antibodies described herein act as inhibitors of KIT activity. phosphorylation of MAPK, phosphorylation of AKT, or phosphorylation of Stat1, Stat3, or Stat5. Therefore, in one embodiment, the present invention can inhibit downstream signaling of The anti-KIT antibodies described herein can be produced by any method described herein or known to those skilled in the art, e.g., by Western blotting. ELISA assay, or immunoblotting assay as described in Section 6. When assessed by ELISA, the effect of KIT ligand stimulation without antibody or with an irrelevant antibody (e.g., phosphorylation of MAPK (e.g., antibodies that do not immunospecifically bind to KIT) compared to phosphorylation in the presence of For example, KIT ligand (e.g., SCF)-induced phosphorylation of MAPK is reduced by at least about 5%, 10%, 15%, or %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, 98%, or 99% inhibition or reduction. The anti-KIT antibodies described herein can be prepared by any of the methods described herein or known to those skilled in the art. For example, Western blot, or the ELISA assay described in Section 6, or immunoblot As assessed by a binding assay, the presence of KIT ligand stimulation, without or with no antibody AKT phosphorylation compared to that in the presence of a non-relevant antibody (e.g., an antibody that does not immunospecifically bind to KIT) phosphorylation of AKT (e.g., KIT ligand (e.g., SCF)-induced phosphorylation of AKT) for at least about 5 min. %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95%, 98%, or 99% inhibition or reduction. In certain embodiments, the anti-KIT antibodies described herein are those described herein or available from the art. ELISA assays, such as Western blots, or the ELISA assays described in Section 6. or in the presence of KIT ligand stimulation as assessed by immunoblotting assay. Phospholipase A in the absence of antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not immunospecifically bind to KIT) phosphorylation of Stat3 (e.g., KIT ligand (e.g., SCF)-induced phosphorylation of Stat3) compared with (concentration) 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% inhibition or reduction In certain embodiments, the anti-KIT antibodies described herein can be The method may be performed by any method described in the literature or known to those skilled in the art, such as Western blot or the method described in Section 6. The KIT linker was not detected as a marker when assessed by ELISA assays or immunoblotting assays. In the presence of a signal stimulus, no antibody or an irrelevant antibody (e.g., one that does not immunospecifically bind to KIT) phosphorylation of Stat1 or Stat5 (e.g., KIT ligand (e.g., antibody)) compared to phosphorylation in the presence of For example, SCF (SCF-induced phosphorylation) can be reduced 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 can be inhibited or reduced by 99%.

[0305] In some embodiments, the compounds described herein that can act as inhibitors of KIT activity or activity. The described anti-KIT antibodies inhibit KIT-expressing cells (e.g., cancer cells, e.g., TF-1 cells), as well as KIT-expressing cells (e.g., cancer cells, e.g., TF-1 cells). cells that respond to T signaling (e.g., in response to KIT ligand stimulation and KIT signaling) Cell proliferation assays can be performed using methods described in the art. For example, cell proliferation can be regulated by bromodeoxyuridine dichloride (BDO) or bromodeoxyuridine dichloride (BDO). BrdU incorporation (e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369; See Campana et al., 1988, J. Immunol. Meth. 107:79) or (3H)thymidine incorporation (e.g., Blechman et al., Cell, 1995, 80:103-113; Chen, J., 1996, Oncog ene 13:1395-403; see Jeoung, J., 1995, J. Biol. Chem. 270:18367 73). by determining the time interval or by direct measurement at various time intervals (e.g., 12- or 24-hour intervals). by cell count or by the expression of known genes, e.g., proto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, D1, D2, D3, E, etc.) Such proteins can be assayed by detecting changes in their activity. The quality and level of mRNA and activity can be determined by any method known in the art. For example, proteins can be detected by ELISA, Western blotting, or other methods using antibodies, including commercially available antibodies. The mRNA can be quantified by known immunodiagnostic methods such as immunoprecipitation or immunoblotting. The present invention can be carried out using methods that are well known and routine in the art, for example, Northern analysis, RNA analysis, and the like. Quantitation can be achieved using enzyme protection, or polymerase chain reaction in conjunction with reverse transcription.

[0306] In a specific embodiment, the antibodies described herein specifically bind to KIT and When assessed by methods described in the literature or known to those skilled in the art (e.g., BrdU incorporation assay) , cell proliferation by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 5 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% inhibition In a specific embodiment, the antibodies described herein specifically bind to KIT and As assessed by methods described herein or known to those skilled in the art (e.g., BrdU incorporation assay), The cell proliferation is increased 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. 5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60 70-fold, 80-fold, 90-fold, or 100-fold inhibition.

[0307] In some embodiments, an anti-KIT antibody described herein that can act as an inhibitor of KIT activity. IT antibodies target cells that express KIT, as well as cells that respond to KIT signaling (e.g., KIT ligases). and reducing or inhibiting the survival of cells that proliferate in response to oxidative stress and KIT signaling. Cell viability assays are described in the art and can be easily performed by one of skill in the art. For example, cell viability can be assessed using trypan blue staining or other cell viability assays known in the art. In a specific embodiment, the level of cellular ATP can be assessed using a cell viability marker. In a specific embodiment, cell viability is determined by measuring the level of Assays standard in the art for measuring intracellular ATP levels, such as the CellTiter-Glo assay, are used. The decrease in cellular ATP is measured at 3 and 7 days using Seikit (Promega). In another specific embodiment, cell viability is measured by neutral red uptake. In another embodiment, the morphological changes can be measured by visual observation. Enlargement, granularity, cells with jagged edges, lamellar appearance, rounding, detachment from the well surface These changes may include cytotoxicity, cytotoxicity, or other changes. These changes may be determined according to the degree of cytotoxicity observed. T (100% toxicity), PVH (partial toxicity - very strong - 80%), PH (partial toxicity - strong - 60%), P ( They are given the designations Ps (partial toxicity - 40%), Ps (partial toxicity - only - 20%), or 0 (no toxicity - 0%). The 50% cell inhibition (cytotoxicity) concentration (IC 50 ) is determined by regression analysis of these data. It is determined.

[0308] In a specific embodiment, the antibodies described herein specifically bind to KIT and Assessed by methods described in the literature or known to those skilled in the art (e.g., trypan blue exclusion assay). When the cells (e.g., cancer cells) are cultured, cell (e.g., cancer cell) survival is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 1 0%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 %, 98%, or 99% inhibition (e.g., partial inhibition). The antibodies described herein specifically bind to KIT and are capable of binding to any of the antibodies described herein or known to those of skill in the art. cell (e.g., cancer cell) viability as assessed by methods (e.g., trypan blue assay) The amount of the present invention is at least about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, Inhibits 80, 90, or 100 fold.

[0309] In some embodiments, an anti-KIT antibody described herein that can act as an inhibitor of KIT activity. The IT antibody is capable of detecting KIT-expressing cells (e.g., cancer cells, e.g., MO7E cells) as well as KIT signaling. cells that respond to transduction (e.g., cells that proliferate in response to KIT ligand stimulation and KIT signaling) It can induce apoptosis (i.e., programmed cell death) in cells. Such systems have been described in the art and can be readily performed by those skilled in the art. Using cytometry, we have been able to measure the activity of apoptosis-mediating enzymes in cells undergoing apoptosis. Activated caspase 3 can be detected using Western blotting or Cleavage of poly(ADP-ribose) polymerase (PARP) can be detected (e.g., Smolich (See, e.g., Wang et al., Blood, 2001, 97:1413-1421). Cleavage of PARP is a key factor in the progression of apoptosis. In a specific embodiment, the antibodies described herein specifically bind to KIT. and methods described herein or known to those skilled in the art (e.g., for detecting activated caspase 3). Apoptosis was reduced to at least about 5%, as assessed by flow cytometry (for cell death). 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 98%, or 99% induction or enhancement. The antibodies described herein specifically bind to KIT and are known to those skilled in the art as described herein or as antibodies to KIT. By known methods (e.g., flow cytometry to detect activated caspase 3), When assessed by the , 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x , 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold induction or enhancement.

[0310] In some embodiments, an anti-KIT antibody described herein that can act as an inhibitor of KIT activity. IT antibodies can be measured by methods commonly known in the art, for example, by soft agar assay. When the cells expressing KIT (e.g., H526 cells or CHO cells expressing exogenous KIT), and cells that respond to KIT signaling (e.g., cells that respond to KIT ligand stimulation and KIT signaling). inhibit anchorage-independent cell growth (e.g., colony formation) by cells that grow in response to In a specific embodiment, the antibodies (or The antigen-binding fragment thereof, or a conjugate thereof) specifically binds to KIT and is described herein. Scaffold non-associated proteins as assessed by methods described or known to those skilled in the art (e.g., soft agar assay). Inhibit cell growth 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% inhibition In a specific embodiment, the antibodies described herein (or their The antigen-binding fragment or conjugate thereof specifically binds to KIT and is described herein. or anchorage-independent as assessed by methods known to those skilled in the art (e.g., soft agar assay). inhibits or reduces sexual cell growth by at least about 25% or 35%, optionally by up to about 75%. In a specific embodiment, the antibodies described herein specifically bind to KIT and are Scaffold non-associated proteins as assessed by methods described or known to those skilled in the art (e.g., soft agar assay). Inhibits dependent cell growth 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, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, Inhibit or reduce by 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0311] Suitable cells and cell lines for use in the assays described herein for KIT activity include: They are readily available (e.g., from the ATCC) or can be readily identified using methods known in the art. For example, a cell that endogenously expresses KIT or that inhibits KIT signaling or activity can be identified. Cells and / or cell lines harboring the present invention are known to those skilled in the art. Suitable cells or cell lines for use in the assays described herein may be endogenously or recombinantly derived. In certain embodiments, the KIT can be expressed in cell proliferation assays. The cells or cell lines for this purpose express KIT endogenously or recombinantly and are capable of expressing KIT ligands (e.g., They are capable of proliferating or increasing proliferation in response to stimulation with, for example, SCF. Cells or cell lines for use in the assay may express KIT endogenously or recombinantly. , which can result in changes in cell viability in response to KIT ligand (e.g., SCF) stimulation Cells or cell lines for use in apoptosis assays may be endogenously or recombinantly derived. They express KIT and undergo apoptotic changes in response to stimulation with KIT ligands (e.g., SCF). It can be done.

[0312] Non-limiting examples of cells that can be used in the methods and assays described herein include: Primary cells, cancer cells, transformed cells, stem cells, mast cells, primordial germ cells, oocytes, sperm Mother cells, embryonic stem cells, hematopoietic cells, erythroleukemia cells (e.g., F36P and TF-1 cell lines), human bone marrow leukemia cell lines, such as MO7E cells; gastrointestinal stromal tumor cell lines, such as ST-882, GIST-T1; GIST48, GIST48B, GIST430, and GIST882; neuroblastoma cell lines, e.g., SK-N-SH, SK-SY5Y; H-EP1, SK-N-BE(2), SK-N-BE(ZkM17), SK-N-BE(2)C, LA-N-1, or LA-N-1-5s; Ewing Sarcoma cell lines, such as TC71, TC32, RD-ES, 5838, A4573, EWS-925, NCI-EWS-94, and NCI -EWS-95; and small cell lung cancer cell lines, such as H526, ECC12, TMK1, MKN7, GCIY, and HGC27. Examples include:

[0313] Alternatively, cells and cell lines expressing KIT, e.g., human KIT, can be routinely produced recombinantly. The following list includes a non-limiting list of cells that can be engineered to recombinantly express KIT: Typical examples include COS cells, HEK 293 cells, CHO cells, fibroblasts (e.g., human fibroblasts), and In a specific embodiment, the present invention provides a method for producing a medicament for the treatment of dengue fever, such as bronchitis, bronchitis, and bronchitis. Cells for use in the methods described herein include CHO cells, such as those available from the CHO GS System™ (Lonza In certain embodiments, these modified cells are CHO cells derived from full-length human KIT (e.g., For example, exogenously express a gene (e.g., SEQ ID NO: 1).

[0314] In some embodiments, an anti-KIT antibody described herein that can act as an inhibitor of KIT activity. IT antibodies inhibit tumor growth or induce tumor regression in mouse model studies For example, tumor cell lines can be introduced into nude mice, and the mice can then be The anti-KIT antibodies described herein can be administered one or more times, and the tumor progression of the injected tumor cells can be monitored. can be monitored over a period of weeks and / or months. The administration of anti-KIT antibodies to nude mice can be carried out before the introduction of tumor cell lines. Suitable tumor cell lines (e.g., tumor cell lines expressing KIT) are transfected into mice using the xenotransplantation method described herein. These tumor xenograft mouse models can be used. Non-limiting examples of cell lines include megakaryoblastic leukemia cell lines, e.g., MO7e; gastrointestinal stromal tumor tumor cell lines, e.g., ST-882, GIST-T1, GIST430, GIST48, GIST48B, and GIST882; human red blood cell lines; Leukemia cell lines, such as HEL and TF-1; human promyelocytic leukemia cell line, HL60; neuroblastoma cells strains, e.g. SK-N-SH, SK-SY5Y, H-EP1, SK-N-BE(2), SK-N-BE(ZkM17), SK-N-BE(2)C, LA -N-1, or LA-N-1-5s; Ewing's sarcoma cell lines, e.g., TC71, TC32, RD-ES, 5838, A4573 , EWS-925, NCI-EWS-94, and NCI-EWS-95; and small cell lung cancer cell lines, such as H526, DMS1 53, DMS79, ECC12, TMK1, MKN7, GCIY, and HGC27. The tumor cell lines for use in the xenograft mouse model are GIST882, GIST430, and GIST48. , GIST48B, HEL, HL60, H526, DMS153, or DMS79 cell line. Suitable cell lines for use in xenograft tumor models recombinantly express KIT. In a specific embodiment, the antibodies described herein can be prepared by The antibody (or an antigen-binding fragment thereof, or a conjugate thereof) specifically binds to KIT. in a mouse model as assessed by methods described herein or known to those skilled in the art. and inhibiting tumor growth 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% Inhibit or inhibit tumor regression by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% ,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%,98%, In a specific embodiment, the antibodies described herein (or their equivalents) induce 99% or more of the IL-16-positive cells. The antigen-binding fragment of the antibody or a conjugate thereof specifically binds to KIT and is a conjugate of the antibody or a conjugate thereof. inhibited tumor growth in mouse models as assessed by methods described above or known to those skilled in the art. or inhibit tumor regression by at least about 25% or 35%, optionally up to about 75%. In a specific embodiment, the method of the present invention induces up to about 25% or 35%, and optionally up to about 75%. The antibodies described herein specifically bind to KIT and can be isolated by methods described herein or known to those of skill in the art. Therefore, when evaluated, tumor growth was reduced by at least about 1-fold, 1.2-fold, or 1. 3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold inhibition or tumor regression at least about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, Induction of tumor growth by 60, 70, 80, 90, or 100 times. Tumor growth inhibition or tumor regression is determined by over a period of time, e.g., by physical measurement of palpable tumors or other visual detection methods. For example, tumor size can be assessed by monitoring tumor size. The cell lines may be engineered to express a visualization agent, such as green fluorescent protein (GFP) or luciferase. can be engineered to express GFP, and then visualized in vivo by microscopy. In vivo visualization of luciferase can be performed by xenografting luciferase substrates The luciferase enzyme, which processes the luciferase substrate, is administered to mice. This can be done by detecting light. The degree of detection of GFP or luciferase Alternatively, the levels correlate with tumor size in xenografted mice.

[0315] In some embodiments, the anti-KIT antibodies described herein specifically bind to the KIT antigen and inhibit tumor differentiation. In a seed transplant model, animal survival can be increased. Therefore, the antibodies (or antigen-binding fragments thereof, or conjugates thereof) described herein and specifically bind to KIT, as assessed by methods described herein or known to those skilled in the art. and in tumor xenograft models, mouse survival is increased by at least about 5%, 10%, 15%, 20%, or %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 98%, or 99%. The antibody (or antigen-binding fragment thereof, or conjugate thereof) specifically binds to KIT. and tumor xenograft models as assessed by methods described herein or known to those of skill in the art. In the present invention, mouse survival is increased by at least about 25% or 35%, and optionally by about 75%. In a specific embodiment, the antibodies described herein specifically bind to KIT and in tumor xenograft models as assessed by methods described herein or known to those skilled in the art. and increase mouse survival by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold. , 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x , 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold increase in survival. Survival is measured by a survival curve of the number of surviving mice. The IL-10 expression level is determined by plotting the IL-10 expression level against time (e.g., days or weeks) after injection of the tumor cell line. It is possible.

[0316] Provided herein are methods for isolating KIT polypeptides, e.g., human KIT polypeptides, with specific affinities. A peptide, such as an antibody, that immunospecifically binds to the D4 region of KIT, eg, human KIT.

[0317] The "affinity" of an antibody described herein for an epitope (e.g., a KIT epitope) is determined by: A term well understood in the art that describes the degree of binding of an antibody to an epitope. Affinity can be measured and / or expressed in several ways known in the art. Such methods include, but are not limited to, measuring the equilibrium dissociation constant (K D or K d ), apparent Equilibrium dissociation constant (K D ' or K d '), and IC 50 (required to achieve 50% inhibition in a competitive assay) For purposes herein, affinity refers to the amount of antibody that binds to an epitope. It is understood that the affinity is the average affinity of a given antibody population combined. ) K as described herein in units of Ig or mg / mL D The values ​​of ' indicate mg Ig per mL of serum. Alternatively, plasma may be used. Antibody affinity may be assessed by administering the therapeutic methods described herein or by administering the therapeutic methods described herein. When used as the basis for the selection of the above treatments, antibody affinity should be measured before and / or during treatment. The resulting values ​​can be used to assess whether a human patient is an appropriate candidate for treatment. It can be used by clinicians when

[0318] In a specific embodiment, provided herein is a KIT antigen, preferably a human KIT antigen. In particular, an antibody (or its antigen) having high binding affinity to the D4 / D5 region of human KIT binding fragments, or conjugates thereof) (e.g., 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, or less than 50 pM K D (Antibody having

[0319] In a specific embodiment, an antibody (or antigen-binding fragment thereof) described herein is and their conjugates) are immunogenic to KIT antigens (e.g., the D4 / D5 region of KIT, e.g., human KIT). and immunospecifically bind to the antibody, as determined by the assays described herein or known to those skilled in the art (e.g., BIA). Evaluated using the Biacore™ assay (Biacore™ International AB, Uppsala, Sweden) When the concentration was less than 500,000pM (500nM), less than 100,000pM (100nM), less than 50,000pM (50nM), less than 10,000 pM (less than 10nM), less than 3,000pM (3nM), less than 2,500pM (2.5nM), less than 2,000pM, less than 1,500pM, 1, Less than 000pM, Less than 750pM, Less than 500pM, Less than 250pM, Less than 200pM, Less than 150pM, Less than 100pM, 75p A dissociation constant (K D In a specific embodiment, the antibodies described herein (if or an antigen-binding fragment thereof, or a conjugate thereof) is a KIT antigen (e.g., KIT, e.g., and the D4 / D5 region of human KIT), and Assays using methods such as the Biacore™ assay and KinExA 3000 instrument When the concentration was 25-100,000pM, 25-75,000pM, 25-50,000pM, 25-40,000pM, 25-30,000pM, 5 to 20,000 pM, 25 to 10,000 pM, 25 to 1,000 pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM, or 25 K in the range of ~50 pM D In certain embodiments, the antibodies (or The antigen-binding fragment thereof, or a conjugate thereof) binds to a KIT antigen (e.g., KIT, e.g., human and immunospecifically binds to the D4 region of human KIT using methods described herein or known to those of skill in the art (e.g., When assessed using a Biacore™ assay, e.g., an assay using a KinExA 3000 instrument , about 100 pM to about 250 nM, or any value therebetween D It has.

[0320] In a specific embodiment, an anti-KIT antibody (or an antigen-binding fragment thereof, or a component thereof) adjugate) immunospecifically binds to a KIT antigen (e.g., the D4 region of KIT, e.g., human KIT). , using assays described herein or known to those skilled in the art (e.g., assays described in Section 6 herein). <3000 pM (3 nM) and <2500 pM for the antigen as assessed using a solid-phase ELISA (as described). Less than M (2.5nM), less than 2000pM, less than 1500pM, less than 1000pM, less than 750pM, less than 500pM, less than 250pM The concentration at 50% binding is less than 200 pM, less than 150 pM, less than 100 pM, or less than 75 pM. In an embodiment, the antibodies (or antigen-binding fragments thereof, or their derivatives) described herein are conjugate) immunospecifically binds to a KIT antigen (e.g., KIT, e.g., the D4 region of human KIT). and methods described herein or known to those skilled in the art (e.g., those described in Section 6 herein). When evaluated using a solid-phase ELISA (using a conventional antibody), the antibody had a response rate of 25-500,000 pM (500 nM) and 25-250,000 pM (500 nM) to the antigen. 0pM (250nM), 25~100,000pM (100nM), 25~75,000pM (75nM), 25~50,000pM (50nM), 25~40 ,000pM(40nM), 25~30,000pM(30nM), 25~20,000pM(20nM), 25~10,000pM(10nM), 25~1, 50% binding concentration in the range of 000pM (1nM), 25-500pM, 25-250pM, 25-100pM, or 25-50pM In certain embodiments, the antibodies (or antigen-binding fragments thereof) described herein or a conjugate thereof) binds to a KIT antigen (e.g., the D4 region of KIT, e.g., human KIT). immunospecifically binds to and is capable of binding ... When assessed using the solid-phase ELISA described in Section 6), the antibody titer against the antigen ranged from about 1 nM to about 25 nM. In certain embodiments, the concentration at 50% binding is The antibody (or antigen-binding fragment thereof, or conjugate thereof) described in For example, the D4 region of KIT (e.g., human KIT) and Evaluated using methods known to those skilled in the art (e.g., solid-phase ELISA, as described in Section 6 herein). When the antibody is tested, the concentration at 50% binding to the antigen is about 50 pM to about 500 pM, or any value therebetween. In certain embodiments, the antibodies (or antigen-binding fragments thereof) described herein or a conjugate thereof) is a nucleotide sequence that is immunized to a KIT antigen (e.g., the D4 region of KIT, e.g., human KIT). and immunospecifically binds to the antibody, and the antibody is capable of being isolated by methods described herein or known to those skilled in the art (e.g., see Section 6 of the present specification). When evaluated using the solid-phase ELISA described in Section 2.1, the concentrations were approximately 0.5 nM, 0.25 nM, 0.1 nM, 1 nM, and 1 .5nM, 2nM, 2.5nM, 3nM, 3.5nM, 4nM, 4.5nM, 5nM, 5.5nM, 6nM, 6.5nM, 7nM, 8nM, 9nM , 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, In certain embodiments, the concentration at 50% binding is 500 nM or less. The antibodies described herein are immunospecific for a KIT antigen (e.g., the D4 region of KIT, e.g., human KIT). and can be measured by methods described herein or known to those skilled in the art (e.g., ELISA, KinExA 3000 instrument). or Biacore™ assay), The concentration at 50% binding of M.

[0321] Methods for determining the affinity of an antibody for its target antigen are readily available and readily available. For example, the affinity and binding properties of an antibody for its target antigen are described in Various in vitro assays known in the art (biochemical or immunological based assays) can be used. assay), e.g., equilibrium assays (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay RIA), or kinetic (e.g., Biacore™ analysis), as well as other methods, e.g., Tight binding assay, competitive inhibition assay, fluorescence resonance energy transfer (FRET), immunoprecipitation, gel electrophoresis It can be determined by electrophoresis and chromatography (eg, gel filtration). These and other methods utilize labels on one or more of the components under consideration, and / or Various detection methods may be utilized, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labeling. In some embodiments, the use of a label is not necessary, for example, The Biacore™ system utilizes a label-free, natural phenomenon called surface plasmon resonance (SPR). A detailed description of binding affinity and kinetics is available in Focusing on antibody-immunogen interactions, Paul, W.E. (ed.), Fundamental Immunology The text can be found in the Journal of the American Psychological Association, 4th ed. (Lippincott-Raven, Philadelphia 1999).

[0322] In some embodiments, a KIT antigen, e.g., human KIT, e.g., the D4 region of KIT (e.g., human KIT), is The affinity of the antibodies described herein for the target region can be indirectly determined using cell-based assays. For example, cells expressing KIT on their cell membrane surface can be identified by immunoblotting with an anti-KIT antibody. and a cellular activity downstream of KIT can be measured using assays known in the art. For example, phosphorylation of the cytoplasmic domain of KIT can be determined by treating cells with an anti-KIT antibody. After contact, the activity is determined by immunoblotting (or Western blotting). Cell extracts can be obtained and specific phosphotyrosine in the cytoplasmic domain of KIT can be detected. Immunoblotting with antibodies that bind but not non-phosphorylated tyrosine (e.g., Cell extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins separated on the gel are transferred to a membrane (e.g., nitrocellulose or polyvinylidene fluoride). The specimen is then processed for transfer to PVDF.

[0323] In certain embodiments, the anti-KIT antibodies described herein target a KIT antigen, e.g., human KIT, For example, it specifically binds to the D4 region of KIT (e.g., human KIT) in the presence of the KIT ligand SCF or In some embodiments, the dimerization and phosphorylation of KIT is induced or enhanced in the absence of In this regard, the anti-KIT antibodies described herein inhibit the binding of a KIT ligand, e.g., SCF, to KIT. The anti-KIT antibody can inhibit or reduce binding to KIT (i.e., the anti-KIT antibody inhibits binding to KIT ligands). In such cases, the cells are treated with an anti-KIT antibody and a KIT linker. The extent of inhibition of KIT phosphorylation can be measured by measuring binding to KIT and KIT linker. The level of anti-KIT antibody that competes with the antibody can be determined.

[0324] Antibodies include monoclonal antibodies, recombinantly produced antibodies, and multispecific antibodies (bispecific antibodies). antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, two heavy chain molecules and two light chain molecules Tetrameric antibody molecules, including antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, and antibody heavy chain dimers. dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies Monospecific antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs) (e.g., monospecific, bispecific, etc.) including camelized antibodies, affibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(s) dFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and any of the above. In one embodiment, the present invention provides an epitope-binding fragment. The term "antibody" as used herein refers to a polyclonal antibody population. Any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., , IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2 In certain embodiments, the antibodies described herein can be of the IgG1a or IgG2b subtype. The antibody may be an IgG antibody, or any of its classes (e.g., human IgG1 or IgG4) or subclasses. In a specific embodiment, a monoclonal antibody is derived from a single hybridoma or other and antibodies produced by a cell of the present invention, wherein the antibodies are detected by, for example, ELISA or Other antigen binding or competition known in the art or in the examples provided herein Immunospecifically binds to the D4 region of the human KIT epitope as determined by binding assays The term "monoclonal" is not limited to any particular method of producing the antibody.

[0325] In certain embodiments, the antibodies provided herein bind to a KIT polypeptide, e.g., K In a specific embodiment, the antibody fragments described herein are Fab fragments that immunospecifically bind to the D4 region of the IT. The antibodies described herein are monoclonal antibodies or isolated monoclonal antibodies. In a specific embodiment, the antibodies described herein are humanized monoclonal antibodies. In certain embodiments, the antibodies described herein are recombinant antibodies, e.g., recombinant human antibodies. In one embodiment, the antibody is a recombinant humanized antibody, or a recombinant monoclonal antibody. The antibodies described herein may contain non-human amino acid sequences, e.g., non-human CDRs or non-human (e.g., , non-human primate) framework residues.

[0326] In certain embodiments provided herein, the recombinant antibody is produced solely by recombinant means. The vector can be isolated, prepared, expressed, or produced, for example, by transfection into a host cell. Antibodies expressed using transfected recombinant expression vectors, recombinant combinatorial antibodies isolated from antibody libraries, or, e.g., synthetic, human immunoglobulin sequences Genetic modification of DNA sequences encoding or sequences encoding human immunoglobulins, e.g. For example, splicing of human immunoglobulin gene sequences into other such sequences can result in Antibodies prepared, expressed, produced, or isolated by any other means, including by extraction. In some embodiments, the amino acid sequence of such a recombinant antibody is modified, and thus, amino acids of such antibodies, e.g., VH and / or VL regions The sequences may be derived from an organism's in vivo antibody germline repertoire, e.g., mouse or human germline In certain embodiments, the recombinant antibody is a sequence that does not naturally occur in the repertoire. , collecting several sequence fragments that are naturally occurring in an organism (e.g., a primate, e.g., a human), The recombinant antibody can be obtained by making the synthetic sequence, in which case the synthetic sequence is It does not occur naturally in organisms (e.g., primates, e.g., humans).

[0327] Antibodies provided herein include any type of immunoglobulin molecule (e.g., IgG IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and and IgA2), or subclasses of immunoglobulin molecules. Therefore, the antibodies provided herein may be IgG antibodies (e.g., human IgG antibodies), or any of a class thereof (e.g., In another specific embodiment, the antibody is a human IgG1 or IgG4 antibody or subclass. The antibodies described herein may be IgG1 (e.g., human IgG1 (isotype a, z, or f)) or I In certain embodiments, the antibodies described herein are whole or partially Entire antibodies, e.g., whole or entire humanized, human, or synthetic human antibodies is.

[0328] The antibodies provided herein target antigens, e.g., KIT epitopes (e.g., the D4 region of human KIT). and an antibody that retains the ability to specifically bind to a KIT epitope in a KIT polypeptide containing the KIT region. In a specific embodiment, the fragments include Fab fragments (antigen-binding domains). It contains a light chain and a portion of a heavy chain (i.e., a heavy chain) cross-linked by a disulfide bond. antibody fragments containing the VH and CH1 domains of the Fab chains); Fab' (Fab and further fragments up to the hinge region); antibody fragments containing a single antigen-binding domain comprising a heavy chain portion containing the antigen-binding domain; F(ab')2 (antibody fragments containing the antigen-binding domain of the heavy chain); two Fab' molecules connected by an interchain disulfide bond in the dimeric region; bispecific Fab (each of which may be directed against a different epitope); a Fab molecule having two antigen-binding domains that can be directed against a specific antigen-binding target; a variable region containing Single Fab chains, also known as sFv (single fragments of an antibody linked by a chain of 10-25 amino acids) variable antigen-binding determining regions of the heavy and heavy chains; disulfide-linked Fv, or dsFv (disulfide-linked the variable antigen-binding determining regions of a single light and heavy chain of an antibody linked by a camel VH (Some amino acids in the VH interface are found in the heavy chains of natural camel antibodies.) the variable antigen-binding determining region of a single heavy chain of the antibody; bispecific sFv (each of which binds to a different epitope); sFv or dsFv molecules with two antigen-binding domains that can be directed against each other; diabodies ( The VH domain of the first sFv associates with the VL domain of the second sFv, and the VL domain of the first sFv associates with the VL domain of the second sFv. Dimerized sFv formed when the VH domains of two sFvs associate; two antigens of a diabody The binding regions may be for the same or different epitopes); and triabodies ( Formed in a manner similar to diabodies, but with three antigen-binding domains in a single complex The resulting trimerized sFv; the three antigen-binding domains may be directed against the same or different epitopes. The antibodies provided herein also include one or more CDR sequences of the antibody. When two or more CDR sequences are present, the CDR sequences can be linked on the scaffold. In certain embodiments, the antibody comprises a single chain Fv ("scFv"). , an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are bound to a single Typically, scFv polypeptides are composed of a VH domain and a VL domain. and a polypeptide linker, which linker is capable of binding the scFv to a desired target site for antigen binding. For a review of scFvs, see Monoclonal Antibody Pharmacology (T The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Spring See Pluckthun, 1994, Er-Verlag, New York, pp. 269-315. Without being bound by any particular theory, it is believed that Fv molecules, due to their small size, are less likely to penetrate tissues. Intact antibodies can be enzymatically cleaved with pepsin to produce F(ab')2 fragments. or can be enzymatically cleaved with papain to produce two Fab fragments. It can be done.

[0329] In certain embodiments, the antibodies described herein are human, synthetic human, or humanized monoclonal antibodies. In certain embodiments, the antibodies described herein are engineered antibodies, e.g., monoclonal antibodies. For example, recombinantly produced antibodies. The antibodies described herein may comprise one or more non-human (e.g., rodent or murine) CDRs and one or more human frameworks. a humanized antibody comprising a human heavy chain constant region (FR), and optionally a human heavy chain constant region and / or a human light chain constant region; In specific embodiments, the antibodies described herein are derived from one or more primates (or non-primates). In a specific embodiment, the nucleotide sequences described herein include a human primate (human primate) framework region. The antibody does not comprise a non-human primate framework region.

[0330] Antibodies provided herein include antibodies that contain chemical modifications, e.g., antibodies that have been chemically modified, e.g., Antibodies that have been modified by the covalent attachment of any type of molecule to the body may be included, for example. The anti-KIT antibody may be, but is not limited to, glycosylated, acetylated, pegylated, phosphate-modified, or the like. can be modified or amidated and can be derivatized with protecting / blocking groups or may further comprise a cellular ligand and / or other proteins or peptides, etc. For example, the antibodies provided herein can be modified, e.g., by glycosylation, acetylation, or the like. , PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein Chemical modification by proteolytic cleavage, binding to cellular ligands or other proteins, etc. Additionally, the anti-KIT antibodies described herein may contain one or more non-classical amino acids. It can have.

[0331] In certain embodiments, provided herein are large-scale manufacturing, e.g., Lonza (Ba The anti-KIT antibody is modified in a manner suitable for the manufacturing platform of the company (Sel, Switzerland). For example, The BI-HEX® technology platform (Boehringer Ingleheim, Germany) was used to The anti-KIT antibodies can be adapted for suitable large-scale production in recombinant mammalian cell expression systems. Such adaptations may involve altering the coding sequence of a desired domain of the anti-KIT antibody, such as one or more CDRs or FRs. The method may include cloning the polynucleotide sequence to be encoded into a suitable expression vector, The vector contains a polynucleotide encoding a suitable constant region so that a complete antibody can be produced. The polynucleotide sequence provided by the expression vector is capable of expressing the gene in a cell. Culture production conditions and purification processes were optimized to maximize antibody yield and stability. It is a nucleotide sequence that can be optimized.

[0332] (5.1.1. Conjugates) In some embodiments, provided herein are diagnostic, detection, or Antibodies conjugated or recombinantly fused to a therapeutic agent or any other molecule (e.g. Conjugated antibodies are antibodies, e.g., human or humanized antibodies, or antigen-binding fragments thereof. or recombinant fused antibodies, for example, for preventing the onset, development, progression, or progression of a KIT-associated disorder or disease. and / or severity as part of clinical trial procedures, e.g., to determine the efficacy of a particular therapy. Conjugated or recombinant The fused antibodies can be used, for example, to treat or manage KIT-associated disorders (e.g., cancer). or for treating or managing the effects of a KIT-associated disorder (e.g., cancer). The antibodies described herein may be characterized by one or more biological and / or molecular properties of the antibody, e.g., Molecules that may affect stability (e.g., in serum), half-life, solubility, and antigenicity (e.g., , polyethylene glycol).

[0333] In certain aspects, provided herein is an antibody (or antibody thereof) described herein. a conjugate comprising a drug (e.g., a therapeutic agent) bound to an antibody (e.g., an antibody-binding fragment), In a specific embodiment, the antibody binds immunospecifically to the D4 region of human KIT (e.g., SEQ ID NO: 15). In this case, the conjugated antibody specifically binds to the D4 region of KIT (e.g., human KIT) and is In a specific embodiment, the conjugate comprises an antibody comprising the CDRs shown in Table 2 or Table 3. The antibody specifically binds to the D4 region of KIT (e.g., human KIT) and is represented by SEQ ID NO: 19, a VL chain region including VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of 20 and 21, and and / or VH CDR1, VH CDR2 having the amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively. and a VH chain region comprising a VH CDR3. specifically binds to the D4 region of KIT (for example, human KIT), and is selected from the group consisting of antibodies Hum1 to Hum20. In specific embodiments, the conjugated antibodies provided herein include those that bind to KIT (e.g., For example, a CDR and a CDR selected from Table 1, Table 2, or Table 3 that specifically bind to the D4 region of human KIT. In a specific embodiment, the present invention includes an antibody comprising a VL and / or a VH comprising a FR selected from 5A to 5D. In particular, the conjugated antibodies provided herein are directed against the D4 region of KIT (e.g., human KIT). and antibodies comprising a VL comprising SEQ ID NO: 12 and / or a VH comprising SEQ ID NO: 11. In embodiments, the conjugated antibody has an affinity of, for example, an EC50 of about 200 pM or less. In another embodiment, the conjugated antibody is an antibody that binds to the D4 region of human KIT. The antibody is an antibody that inhibits the biological activity of KIT. The conjugates include antibodies and molecules (e.g., therapeutic or drug moieties) described herein. , where the antibody is linked to the molecule directly or via one or more linkers. In certain embodiments, the antibody is covalently conjugated to the molecule. In a specific embodiment, the antibody is non-covalently conjugated to the molecule. Therefore, the antibodies described herein, e.g., antibodies conjugated to a drug, can be used to treat wild-type human KI. In certain embodiments, the antibodies described herein, e.g., conjugated to a drug, bind to T. The gated antibodies identify mutations, e.g., somatic mutations associated with cancer (e.g., GIST), For example, a mutation in exon 9 of human KIT that duplicates the Ala and Tyr residues at positions 502 and 503 It binds to the extracellular domain of human KIT, including the heterologous domain.

[0334] Such diagnosis and detection can be achieved, for example, by coupling an antibody to a detectable molecule or substance. This can be achieved by activating various enzymes, such as limiting enzymes. However, horseradish peroxidase, alkaline phosphatase, and β-galactosidase prosthetic groups, such as, but not limited to, streptavidin; fluorophores, including but not limited to, thiazolinone / biotin and avidin / biotin; Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, di Chlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent substances, such as, but not limited to, luminol; bioluminescent substances, such as, but not limited to, , luciferase, luciferin, and aequorin; radioactive substances, including but not limited to However, iodine ( 131 I, 125 I, 123 I, and 121 I,), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium( 115 In, 113 In, 112 In, and 111 In), technetium ( 99 Tc), thallium ( 201 Ti) ,gallium( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), Sn( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 18 8 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51Cr, 54 Mn, 75 Se, 113 Sn, and 117 Sn; and various positron-emitting metals using positron emission tomography , as well as non-radioactive paramagnetic metal ions.

[0335] Provided are compounds conjugated or combined with a therapeutic moiety (or more than one therapeutic moiety). Recombinantly fused antibodies or antigen-binding fragments thereof described herein, and the use of such antibodies The antibody may be used to inhibit the production of a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent, or a or a radioactive metal ion, e.g., an α-emitter, Cytotoxins or cytotoxic agents include those that are detrimental to cells. Therapeutic moieties include any agent which is an auristatin or a derivative thereof, e.g. For example, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin statin PYE, and auristatin E (AE) (see, e.g., U.S. Pat. No. 7,662,387 and U.S. Pat. No. 5,662,387). See Application Nos. 2008 / 0300192 and 2008 / 0025989, each of which is incorporated herein by reference. incorporated herein); microtubule disrupting agents, such as maytansine or its derivatives, e.g. , maytansinoid DM1 (see, e.g., U.S. Pat. Nos. 7,851,432, 7,575,748, and 5,416, See US Pat. No. 6,064, each of which is incorporated herein by reference; Prodromal drugs, e.g., prodrugs of CC-1065 (rachelmycin) analogs; antimetabolites (e.g., Thotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil alkylating agents (e.g., mechlorethamine, thioepaclorambucil, Lupharan, carmustine (BCNU) and lomustine (CCNU), cyclothosfamide phamide), busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP), and cisplatin); minor group Conjugated alkylating agents; anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin; antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC); auristatin molecules (e.g., For example, auristatin PHE, bryostatin 1, and solastatin 10; Woyke et al., Ant Antimicrob. Agents Chemother. 46:3802-8(2002), Woyke et al., Antimicrob. Agents Ch Emother. 45:3580-4(2001), Mohammad et al., Anticancer Drugs 12:735-40(2001), Wa ll et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999); Mohammad et al., In J. Oncol. 15:367-72 (1999), all of which are incorporated herein by reference. incorporated); hormonal agents (e.g., glucocorticoids, progestins, androgens) , and estrogen), DNA repair enzyme inhibitors (e.g., etoposide or topotecan), kinases enzyme inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer r Res. 8(7):2167-76(2002)); cytotoxic agents (e.g., paclitaxel, cytochalasin B, Gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide , vincristine, vinblastine, colchicine, doxorubicin, daunorubicin Rubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, Tinomycin D, 1-dehydrotestosterone, glucorticoid, procaine tetracaine, lidocaine, propranolol, and puromycin, and These analogs or homologs, as well as those disclosed in U.S. Patent Nos. 6,245,759, 6,399,633, and 6,383,799, No. 0, No. 6,335,156, No. 6,271,242, No. 6,242,196, No. 6,218,410, No. 6,218,372, No. 6,057,300, No. 6,034,053, No. 5,985,877, No. 5,958,769, No. 5,925,376, No. 5,9 No. 22,844, No. 5,911,995, No. 5,872,223, No. 5,863,904, No. 5,840,745, No. 5,728,86 Nos. 5,648,239 and 5,587,459, each of which is a compound disclosed in such a manner. the disclosure of which is incorporated herein by reference); farnesyl tran Spherase inhibitors (e.g., R115777, BMS-214662, and, for example, U.S. Pat. No. 6,458,999) No. 35, No. 6,451,812, No. 6,440,974, No. 6,436,960, No. 6,432,959, No. 6,420,387 , No. 6,414,145, No. 6,410,541, No. 6,410,539, No. 6,403,581, No. 6,399,615, No. 6 ,387,905, No. 6,372,747, No. 6,369,034, No. 6,362,188, No. 6,342,765, No. 6,342, No. 487, No. 6,300,501, No. 6,268,363, No. 6,265,422, No. 6,248,756, No. 6,239,140 , No. 6,232,338, No. 6,228,865, No. 6,228,856, No. 6,225,322, No. 6,218,406, No. 6 ,211,193, No. 6,187,786, No. 6,169,096, No. 6,159,984, No. 6,143,766, No. 6,133, No. 303, No. 6,127,366, No. 6,124,465, No. 6,124,295, No. 6,103,723, No. 6,093,737 , No. 6,090,948, No. 6,080,870, No. 6,077,853, No. 6,071,935, No. 6,066,738, No. 6 Nos. 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305 each of which is incorporated herein by reference for its disclosure of such inhibitors. incorporated herein); topoisomerase inhibitors (e.g., camptothecin; irinotecan SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-6 22; Rubitecan; Pyrazoloacridine; XR-5000; Saitopine; UCE6; UCE1022; TAN-1518A ; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506; and Rebeccamycin ); vulgarein; DNA minor groove binders, such as Hoescht dye 33342 and Hoechst dye Substance 33258; Nitidine; Fagaronine; Epiberberine; Coraline; β-Lapachone; BC-4-1; Bispho Sulfonates (e.g., alendronate, cimadronate, clodronate, Tiludronate, etidronate, ibandronate, neridronate, olpandrone acetate, risedronate, piridronate, pamidronate, zolendronate), HMG-CoA Reductase inhibitors (e.g., lovastatin, simvastatin, atorvastatin, prasugrel) statin, fluvastatin, statin, cerivastatin, Lescol, Lupitor, Rosuva statins, and atorvastatin; antisense oligonucleotides (e.g., U.S. Pat. No. 6,113,239); Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709 Each of the disclosures is incorporated by reference in its entirety with respect to such oligonucleotides. adenosine deaminase inhibitors (e.g., fludara phosphate); ibritumomab tiuxetan (Zevalin®) ); tositumomab (Bexxar®), and pharmaceutically acceptable salts and solvates thereof Examples of compounds include, but are not limited to, compounds, clathrates, and prodrugs. In the present invention, antibodies conjugated to such therapeutic / drug moieties have a specific activity less than about 200 pM of the parent In another embodiment, such a therapeutic agent is an antibody that binds to the D4 region of human KIT. The antibody conjugated to the target / drug moiety is an antibody that inhibits the biological activity of KIT. In a specific embodiment, the antibody conjugated to such a therapeutic / drug moiety is , CDRs shown in Table 1 (e.g., having the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively) VL CDR1, VL CDR2, and VL CDR3, and / or SEQ ID NOs: 16, 17, and 18, respectively. a VH chain region comprising a VH CDR1, a VH CDR2, and a VH CDR3 having an amino acid sequence of In a specific embodiment, the antibody comprises a CDR as set forth in Table 3. Such antibodies conjugated to a therapeutic / drug moiety may be selected from SEQ ID NOs: 7, 8, 9, 10, or 12, or a VL comprising a sequence shown in Tables 5B and 5D, and / or SEQ ID NOs: 2, 3, 4, 5, 6, if or 11, or an antibody comprising a VH comprising a sequence shown in Tables 5A and 5C.

[0336] In certain embodiments, the therapeutic moiety or drug moiety is an antitubulin drug, e.g., Non-limiting examples of auristatins include monomethyl auristatins and derivatives thereof. Monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin PYE, and and auristatin E (AE) (see, e.g., U.S. Pat. No. 7,662,387 and U.S. Pat. No. 5,662,387). See published application nos. 2008 / 0300192 and 2008 / 0025989, each of which is incorporated by reference. In certain embodiments, the therapeutic or drug moiety is tubule disrupting agents, such as maytansine or its derivatives, e.g., maytansinoids DM1 or D M4 (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). The therapeutic moiety or drug moiety may be a prodrug, e.g., a CC-1065 (rachelmycin) analogue. (See, e.g., U.S. Patent Application Publication No. 2008 / 0279868, and PCT International Patent Application Publication No. See published applications WO 2009 / 017394, WO 2010 / 062171, and WO 2007 / 089149; (Each of which is incorporated herein by reference.) In one embodiment, such The antibody conjugated to a suitable therapeutic / drug moiety binds to D4 of human KIT with an affinity of less than about 200 pM. In another embodiment, the antibody is an antibody that binds to such a therapeutic / drug moiety. The antibody to be conjugated is an antibody that inhibits the biological activity of KIT. In the present invention, an antibody (e.g., a human or humanized antibody) conjugated to such a therapeutic / drug moiety is The antibody comprises the CDRs shown in Table 1 (e.g., amino acids 19, 20, and 21, respectively). VL CDR1, VL CDR2, and VL CDR3 having the sequences SEQ ID NO: 16, a VH chain region including VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of 17 and 18, or is an antibody comprising the CDRs shown in Table 2 or the CDRs shown in Table 3. In a specific embodiment, and antibodies (e.g., human or humanized antibodies) conjugated to such therapeutic / drug moieties. The antibody) includes a VL comprising SEQ ID NO: 7, 8, 9, 10, or 12, or a sequence shown in Tables 5B and 5D, as well as and / or SEQ ID NO: 2, 3, 4, 5, 6, or 11, or a sequence shown in Tables 5A and 5C. It is an antibody containing VH.

[0337] In a specific embodiment, the antibody and therapeutic / drug substance are linked by one or more linkers. In another specific embodiment, the antibody and therapeutic / drug substance are conjugated to is directly conjugated.

[0338] In a specific embodiment, a therapeutic agent for conjugation to an antibody described herein is Non-limiting examples of moieties or drug moieties include calicheamicin (e.g., LL-E33288 conjugates), , e.g., gamma-calicheamicin, see e.g., U.S. Pat. No. 4,970,198) and derivatives thereof (e.g., gamma calicheamicin hydrazide derivatives), ozogamicin, duocalmicin and its derivatives (e.g., CC-1065 (NSC 298223) or achiral duocarmycins) analogs (e.g., AS-1-145 or centanamycin), taxanes and their derivatives, and Enediyne and its derivatives (see, for example, PCT International Patent Application Publication No. WO 2009 / 017394) , WO 2010 / 062171, WO 2007 / 089149, WO 2011 / 021146, WO 2008 / 150261, WO 200 Reference was made to WO 6 / 031653, WO 2005 / 089809, WO 2005 / 089807, and WO 2005 / 089808. (See, e.g., U.S. Pat. No. 6,223,999, each of which is incorporated herein by reference in its entirety.) In the present invention, antibodies conjugated to such therapeutic / drug moieties have the CDRs ( For example, VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 20, 21, and 22, respectively. and VL CDR3, and / or VH CDR4 having the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively. In a specific embodiment, the antibody comprises a VH chain region comprising VH CDR1, VH CDR2, and VH CDR3. In such cases, antibodies conjugated to such therapeutic / drug moieties are those represented by SEQ ID NOs: 7, 8, 9, 10, , or 12, or a VL comprising the sequence shown in Tables 5B and 5D, and / or SEQ ID NOs: 2, 3, 4, 5, 6, or 11, or an antibody comprising a VH comprising a sequence shown in Tables 5A and 5C. In embodiments, the antibody and therapeutic agent are conjugated by one or more linkers. In another specific embodiment, the antibody and therapeutic agent are directly conjugated.

[0339] Non-limiting examples of calicheamicins suitable for conjugation to the antibodies described herein include: Examples are, for example, U.S. Patent Nos. 4,671,958; 5,053,394; 5,037,651; 5,079,233; and and 5,108,912; and PCT International Patent Application Publication Nos. WO 2011 / 021146, WO 2008 / 150261, W WO 2006 / 031653, WO 2005 / 089809, WO 2005 / 089807, and WO 2005 / 089808 each of which is incorporated herein by reference for its disclosure of such calicheamicins. In certain embodiments, these compounds are suitable thiols. to form disulfides and simultaneously release fun...

Claims

1. Patent application title: TREATING KIT-ASSOCIATED DISORDERS, COMPRISING ANTIBODIES OR ANTIGEN-BINDING FRAGMENTS THEREOF THAT IMMUNOSPECIFICALLY BIND TO HUMAN KIT A pharmaceutical composition for administering or managing The antibody or antigen-binding fragment thereof, (i) VL CD having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. a light chain variable region ("VL") comprising R1, a VL CDR2, and a VL CDR3; and (ii) VHC having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. A heavy chain variable region ("VH") comprising DR1, VH CDR2, and VH CDR3. Including, The pharmaceutical composition, wherein the VL and VH are non-immunogenic in humans.

2. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a human IgG constant region. 。

3. The pharmaceutical composition of claim 2, wherein the human IgG constant region comprises a non-naturally occurring Fc region.

4. The pharmaceutical composition according to claim 2 or 3, wherein the human IgG constant region comprises one or more polymorphisms in the Fc region. 。

5. The human IgG constant region has a EU interface as shown in Kabat compared to a wild-type human IgG constant region. Index numbered 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 252 、254、256、262、263、264、265、266、267、269、296、297、298、299、313、325、326 , 327, 328, 329, 330, 332, 333, and 334. The pharmaceutical composition according to any one of claims 2 to 4, comprising a polyacid modification.

6. The human IgG constant region has a repeat sequence at positions 252, 254, and 256 compared to a wild-type human IgG constant region. wherein: (a) the amino acid at position 252 is replaced with a tyrosine (Y); (b) the amino acid at position 254 is replaced with a threonine (T); and (c) the amino acid at position 256 is replaced with glutamic acid (E); Any one of claims 2 to 5, wherein the numbering is according to the EU index as set out in Kabat. Item 3. The pharmaceutical composition according to item 2.

7. The KIT-related disorder is selected from the group consisting of gastrointestinal stromal tumors, mastocytosis, inflammatory bowel disease, rheumatoid arthritis, and the like. The pharmaceutical composition according to any one of claims 1 to 6, wherein the inflammation is allergic inflammation.

8. The pharmaceutical composition of claim 7, wherein the KIT-associated disorder is a gastrointestinal stromal tumor.

9. The pharmaceutical composition of claim 7, wherein the KIT-associated disorder is mastocytosis.

10. The pharmaceutical composition of claim 7, wherein the KIT-associated disorder is inflammatory bowel disease.

11. The pharmaceutical composition of claim 7, wherein the KIT-associated disorder is rheumatoid arthritis.

12. The pharmaceutical composition of claim 7, wherein the KIT-associated disorder is allergic inflammation.

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