ANTI-KIT ANTIBODIES AND USES THEREOF
Provided herein, in one aspect, 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 described herein.
1 . An isolated antibody, or an antigen-binding fragment thereof, which immunospecifically binds to a D4 region of human KIT, comprising:
(i) a light chain variable region (“VL”) comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; and
(ii) a heavy chain variable region (“VH”) comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively;
wherein the VL and VH are non-immunogenic in a human.
2 . The antibody of claim 1 , wherein the antibody can be expressed in Chinese hamster ovary (CHO) cells at a titer of at least 0.45 μg/mL.
3 . The antibody of claim 1 , wherein the VL comprises the amino acid sequence of SEQ ID NO: 8, 10, 7, or 9, and the VH comprises the amino acid sequence of SEQ ID NO: 4, 3, 6, 5, or 2.
4 . The antibody of claim 1 , wherein
(i) the VL comprises the amino acid sequence of SEQ ID NO: 8 and the VH comprises the amino acid sequence of SEQ ID NO: 4;
(ii) the VL comprises the amino acid sequence of SEQ ID NO: 10 and the VH comprises the amino acid sequence of SEQ ID NO: 3;
(iii) the VL comprises the amino acid sequence of SEQ ID NO: 8 and the VH comprises the amino acid sequence of SEQ ID NO: 6;
(iv) the VL comprises the amino acid sequence of SEQ ID NO: 7 and the VH comprises the amino acid sequence of SEQ ID NO: 5;
(v) the VL comprises the amino acid sequence of SEQ ID NO: 7 and the VH comprises the amino acid sequence of SEQ ID NO: 2;
(vi) the VL comprises the amino acid sequence of SEQ ID NO: 7 and the VH comprises the amino acid sequence of SEQ ID NO: 3;
(vii) the VL comprises the amino acid sequence of SEQ ID NO: 7 and the VH comprises the amino acid sequence of SEQ ID NO: 4;
(viii) the VL comprises the amino acid sequence of SEQ ID NO: 7 and the VH comprises the amino acid sequence of SEQ ID NO: 6;
(ix) the VL comprises the amino acid sequence of SEQ ID NO: 8 and the VH comprises the amino acid sequence of SEQ ID NO: 2;
(x) the VL comprises the amino acid sequence of SEQ ID NO: 8 and the VH comprises the amino acid sequence of SEQ ID NO: 3;
(xi) the VL comprises the amino acid sequence of SEQ ID NO: 8 and the VH comprises the amino acid sequence of SEQ ID NO: 5;
(xii) the VL comprises the amino acid sequence of SEQ ID NO: 9 and the VH comprises the amino acid sequence of SEQ ID NO: 2;
(xiii) the VL comprises the amino acid sequence of SEQ ID NO: 9 and the VH comprises the amino acid sequence of SEQ ID NO: 3;
(xiv) the VL comprises the amino acid sequence of SEQ ID NO: 9 and the VH comprises the amino acid sequence of SEQ ID NO: 4;
(xv) the VL comprises the amino acid sequence of SEQ ID NO: 9 and the VH comprises the amino acid sequence of SEQ ID NO: 5;
(xvi) the VL comprises the amino acid sequence of SEQ ID NO: 9 and the VH comprises the amino acid sequence of SEQ ID NO: 6;
(xvii) the VL comprises the amino acid sequence of SEQ ID NO: 10 and the VH comprises the amino acid sequence of SEQ ID NO: 2;
(xviii) the VL comprises the amino acid sequence of SEQ ID NO: 10 and the VH comprises the amino acid sequence of SEQ ID NO: 4;
(xix) the VL comprises the amino acid sequence of SEQ ID NO: 10 and the VH comprises the amino acid sequence of SEQ ID NO: 5; or
(xx) the VL comprises the amino acid sequence of SEQ ID NO: 10 and the VH comprises the amino acid sequence of SEQ ID NO: 6.
5 . The antibody of claim 1 , which comprises:
(i) a light chain variable region (“VL”) comprising the amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAPKX K2 LIYSASYRYSG VPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 YX K6 CQQYNSYPRTFGGGTKVEIK, wherein X K1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 is an amino acid with an aliphatic hydroxyl side chain or is P, X K5 is an amino acid with a charged or acidic side chain, and X K6 is an amino acid with an aromatic side chain; and
(ii) a heavy chain variable region (“VH”) comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.
6 . The antibody of claim 1 , which comprises:
(i) (i) a VL comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; and
(ii) (ii) a VH comprising the amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAP GKGLEWIARIYPGSGNTYYNEKFKGRX H4 TX H5 TAX H6 KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS, wherein X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid with an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain.
7 . The antibody of claim 1 , which comprises:
(iii) a VL comprising an amino acid sequence that is: at least 90% identical to SEQ ID NO: 7, at least 88% identical to SEQ ID NO: 8, at least 87% identical to SEQ ID NO: 9, or at least 84% identical to SEQ ID NO: 10; and
(iv) a VH comprising an amino acid sequence that is: at least 93% identical to SEQ ID NO: 2, at least 92% identical to SEQ ID NO: 3, at least 90% identical to SEQ ID NO: 4, at least 87% identical to SEQ ID NO: 5, or at least 86% identical to SEQ ID NO: 6.
8 . An isolated antibody, or an antigen-binding fragment thereof, which immunospecifically binds to a D4 region of human KIT, comprising:
(i) a light chain variable region (“VL”) comprising the amino acid sequence: DIVMTQSPSX K1 LSASVGDRVTITCKASQNVRTNVAWYQQKPGKAP KX K2 LIYSASYRYSGVPDRFX K3 GSGSGTDFTLTISSLQX K4 EDFAX K5 Y X K6 CQQYNSYPRTFGGGTKVEIK, wherein X K1 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K2 is an amino acid with an aromatic or aliphatic hydroxyl side chain, X K3 is an amino acid with an aliphatic hydroxyl side chain, X K4 is an amino acid with an aliphatic hydroxyl side chain or is P, X K5 is an amino acid with a charged or acidic side chain, and X K6 is an amino acid with an aromatic side chain; and
(ii) a VH comprising the amino acid sequence: QVQLVQSGAEX H1 KKPGASVKX H2 SCKASGYTFTDYYINWVX H3 QAP GKGLEWIARIYPGSGNTYYNEKFKGRX H4 TX H5 TAX H6 KSTSTAYMX H7 LSSLRSEDX H8 AVYFCARGVYYFDYWGQGTTVTVSS, wherein X H1 is an amino acid with an aliphatic side chain, X H2 is an amino acid with an aliphatic side chain, X H3 is an amino acid with a polar or basic side chain, X H4 is an amino acid with an aliphatic side chain, X H5 is an amino acid with an aliphatic side chain, X H6 is an amino acid with an acidic side chain, X H7 is an amino acid with an acidic or amide derivative side chain, and X H8 is an amino acid with an aliphatic hydroxyl side chain.
9 . The antibody of claim 8 , wherein X K1 is the amino acid F or S, X K2 is the amino acid A or S, X K3 is the amino acid T or S, X K4 is the amino acid S or P, X K5 is the amino acid D or T, and X K6 is the amino acid F or Y.
10 . The antibody of claim 8 , wherein X H1 is the amino acid L or V, X H2 is the amino acid L or V, X H3 is the amino acid K or R, X H4 is the amino acid V or A, X H5 is the amino acid L or I, X H6 is the amino acid E or D, X H7 is the amino acid Q or E, and X H8 is the amino acid S or T.
11 . The antibody of any one of claims 1 to 10 , wherein the antibody specifically binds to CHO cells recombinantly expressing wild-type KIT with an EC50 of about 150 pM or less as determined by flow cytometry.
12 . The antibody of any one of claims 1 to 10 , wherein the antibody inhibits tyrosine phosphorylation of KIT with an IC50 of about 600 pM or less as determined by ELISA.
13 . The antibody of any one of claims 1 to 10 , which can be expressed in CHO cells at a titer of at least 1.0 μg/mL, or at least 1.1 μg/mL, or at least 1.2 μg/mL.
14 . The antibody of any one of claims 1 to 10 , wherein the antibody further comprises a human kappa light chain constant region and a human gamma heavy chain constant region.
15 . The antibody of any one of claims 1 to 10 , wherein the antibody is a human IgG1 or IgG4 antibody.
16 . The antibody of any one of claims 1 to 10 , which is a monoclonal antibody.
17 . The antibody of any one of claims 1 to 10 , which is an antigen-binding fragment or a Fab fragment.
18 . The antibody of any one of claims 1 to 10 , which is a bispecific antibody.
19 . The antibody of any one of claims 1 to 10 , which is fused to a heterologous polypeptide.
20 . A conjugate comprising the antibody of any one of claims 1 to 10 or a antigen-binding fragment thereof linked to an agent.
21 . The conjugate of claim 20 , wherein the agent is a toxin.
22 . The conjugate of claim 21 , wherein the toxin is abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin.
23 . The conjugate of claim 20 , wherein the conjugate is internalized by a cell.
24 . A pharmaceutical composition comprising the conjugate of claim 20 and a pharmaceutically acceptable carrier.
25 . A pharmaceutical composition comprising the antibody of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.
26 . A polynucleotide comprising nucleotide sequences encoding a VH chain region, a VL chain region, or both a VL chain region and a VH chain region, of an antibody of any one of claims 1 to 10 .
27 . The polynucleotide of claim 26 , which comprises SEQ ID NO: 22, 23, 24, 25, or 26 encoding a VH.
28 . The polynucleotide of claim 26 , which comprises SEQ ID NO: 27, 28, 29, or 30 encoding a VL.
29 . The polynucleotide of claim 26 , which comprises SEQ ID NO: 22, 23, 24, 25, or 26 encoding a VH, and SEQ ID NO: 27, 28, 29, or 30 encoding a VL.
30 . A vector comprising the polynucleotide of claim 26 .
31 . The vector of claim 30 , which is a mammalian expression vector.
32 . A host cell comprising the vector of claim 30 or one or more polynucleotides of claim 26 .
33 . An isolated cell producing an antibody according to any one of claims 1 to 10 .
34 . An isolated cell comprising one or more polynucleotides of claim 26 , wherein the cell can express an antibody which specifically binds to a D4 of human KIT.
35 . A cell comprising the vector of claim 30 .
36 . A kit comprising the antibody of any one of claims 1 to 10 .
37 . A kit comprising the conjugate of claim 20 .
38 . A method for treating or managing a KIT-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody of any one of claims 1 to 10 .
39 . A method for treating or managing a KIT-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate of claim 20 .
40 . The method of claim 38 , wherein the KIT-associated disorder is cancer, an inflammatory condition, or fibrosis.
41 . The method of claim 40 , wherein the cancer is leukemia, chronic myelogenous leukemia, lung cancer, small cell lung cancer, or gastrointestinal stromal tumors.
42 . The method of claim 41 , wherein the cancer is refractory to treatment by a tyrosine kinase inhibitor.
43 . The method of claim 42 , wherein the tyrosine kinase inhibitor is imatinib mesylate or SU11248.
44 . The method of claim 38 , wherein the method further comprises administering a second therapeutic agent.
45 . The method of claim 44 , wherein the second therapeutic agent is a chemotherapeutic agent, tyrosine kinase inhibitor, a histone deacetylase inhibitor, an antibody, or a cytokine.
46 . The method of claim 45 , wherein the tyrosine kinase inhibitor is imatinib mesylate or SU11248.
47 . A method for diagnosing a subject with a KIT-associated disorder comprising contacting cells or a sample obtained from the subject with the antibody of any one of claims 1 to 10 and detecting the expression level of KIT in the cells or the sample.
48 . The method of claim 47 , wherein the antibody is conjugated to a detectable molecule.
49 . The method of claim 48 , wherein the detectable molecule is an enzyme, a fluorescent molecule, a luminescent molecule, or a radioactive molecule.
50 . A method for inhibiting KIT activity in a cell expressing KIT comprising contacting the cell with an effective amount of the antibody of any one of claims 1 to 10 .
51 . A method for inducing or enhancing apoptosis in a cell expressing KIT comprising contacting the cell with an effective amount of the antibody of any one of claims 1 to 10 .
52 . A method for inducing cell differentiation comprising contacting a cell expressing KIT with an effective amount of the antibody of any one of claims 1 to 10 .
53 . The method of claim 52 , wherein the cell is a stem cell.
54 . A method of making an antibody which immunospecifically binds to a D4 region of human comprising culturing, and/or expressing the antibody using, the cell of claim 32 .
55 . The method claim 54 further comprising purifying the antibody obtained from said cell.
56 . An isolated antibody or antigen-binding fragment thereof, which immunospecifically binds to a D4 region of human KIT, wherein said antibody or antigen-binding fragment thereof comprises:
(i) a light chain variable region (“VL”) comprising a VL CDR1, VL CDR2, and VL CDR3 selected from the group set forth in Tables 10-12; and
(ii) a heavy chain variable region (“VH”) comprising VH CDR1, VH CDR2, and VH CDR3 selected from the group set forth in Tables 13-15.
57 . An isolated antibody or antigen-binding fragment thereof, which immunospecifically binds to a D4 region of human KIT, wherein said antibody or antigen-binding fragment thereof comprises:
(i) a light chain variable region (“VL”) comprising a VL CDR1, VL CDR2, and VL CDR3 selected from the group set forth in Tables 2 and 3; and
(ii) a heavy chain variable region (“VH”) comprising VH CDR1, VH CDR2, and VH CDR3 selected from the group set forth in Tables 2 and 3.