WO2015050959A1

WO2015050959A1 - Anti-kit antibodies and methods of use thereof

Info

Publication number: WO2015050959A1
Application number: PCT/US2014/058578
Authority: WO
Inventors: Joseph Schlessinger; Andrey Vasilievich RESHETNYAK; Sachdev Singh SIDHU; Bryce Philip NELSON; Irit Lax; Xiarong SHI
Original assignee: Yale University; Kolltan Pharmaceuticals , Inc.
Priority date: 2013-10-01
Filing date: 2014-10-01
Publication date: 2015-04-09
Also published as: WO2015050959A9; WO2015050959A8

Abstract

The present disclosure provides human antibodies that bind the D4 membrane proximal region of the KIT receptor tyrosine kinase. The present disclosure also provides methods of treating KIT-associated diseases, including cancer, using the antibodies of the disclosure.

Description

ANTI-KIT ANTIBODIES AND METHODS OF USE THEREOF

Statement Regarding Federally Sponsored Research or Development

This invention was made with Government support under contract S10RR026992- 0110 awarded by the National Institutes of Health. The government may have certain rights in the invention.

Related Applications

This application claims priority to U.S. Provisional Application No. 61/977,888, filed on April 10, 2014, and U.S. Provisional Application No. 61/960,939, filed on October 1, 2013, the entire contents of each which are expressly incorporated herein by reference.

Background of the Invention

The receptor tyrosine kinase (RTK) KIT is a transmembrane protein that plays crucial roles in mediating diverse cellular processes including cell differentiation, proliferation, and cell survival among other activities. These processes occur through activation of KIT upon binding by Stem Cell Factor (SCF), a ligand found in both membrane anchored and soluble forms (Jiang et al. (2000) EMBO J 19: 3192-3202; Zhang et al. (2000) Proc Natl Acad Sci USA 97: 7732-7737), in a variety of cell types including hematopoietic stem cells, germ cells, vascular endothelial cells, and interstitial cells of Cajal bodies (Fleischman (1993) Trends Genet 9: 285-290; Huizinga et al. (1995) Nature 373: 347-349). KIT belongs to the type III subfamily of RTKs (Lemmon et al. (2010) Cell 141: 1117-1134), a family composed of an extracellular region that includes five Ig-like domains (designated D1-D5), a single transmembrane domain (TM), a juxtamembrane region (JM), a tyrosine kinase domain split by a kinase insert, and a C terminal tail (Roskoski (2005) Biochem Biophys Res Comm 337: 1-13).

Based on the determination of the crystal structure of the complete extracellular region of KIT before and after ligand stimulation (Yuzawa et al. (2007) Cell 130: 323-334) and the tyrosine kinase domain (Mol et al. (2003) J Biol Chem 278:31461-31464; Mol et al. (2004) J Biol Chem 279: 31655-31663), a mechanism has been proposed whereby activation of KIT is initiated through receptor dimerization. Dimerization of the KIT ectodomain is ligand driven and initiated through high affinity binding of an SCF dimer to the membrane distal Ig-like domains (Dl-3) of the receptor (Lemmon et al. (1997) J Biol Chem 272: 6311- 6317; Liu et al. (2007) EMBO J 26: 891-901). "Cros slinking" of the membrane distal Ig-like domains with their ligand, dramatically increases local concentration of the membrane proximal (D4 and D5) and transmembrane domains to allow weak homotypic interactions that mediate D4-D4 and D5-D5 contacts between neighboring KIT molecules. These homotypic associations promote conformational rearrangements that permit correct association between neighboring cytoplasmic regions of KIT dimers resulting in

autophosphorylation, tyrosine kinase stimulation, recruitment of signaling proteins and cell signaling.

Dysregulation of KIT, by different recurring somatic mutations, has been associated with numerous hematopoietic and other cancers, including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Most reoccurring activating mutations map to the cytoplasmic juxtamembrane (JM) region (exon 11) and to the membrane proximal Ig-like domain - D5 (exon 9) of the extracellular region (Ashman et al. (2012) Exp Opin Inv Drug 22: 1-13; Corless et al. (2011) Nat Rev Cancer 11: 865-878). Currently, initial treatment for GIST patients involves the small molecule kinase inhibitor Gleevec (imatinib) that can efficiently block kinase activity of both JM and D5 mutants of KIT. Unfortunately, most GIST patients develop resistance to the drug within two years of treatment by acquiring additional mutations usually mapped to exons 13 and 14 of KIT (V654A and V670I, respectively). Patients resistant to Gleevec are usually treated with the kinase inhibitor, Sutent (Sunitinib), that can inhibit many of the KIT mutations. However, in addition to Gleevec resistant mutations in exons 13 and 14, kinase domain mutants (exon 17) that are resistant to both Gleevec and Sutent have also been identified in GIST patients (Gajiwala et al. (2009) Proc Natl Acad Sci USA 106: 1542-1547). Accordingly, while treatment of these cancers with tyrosine kinase inhibitors initially show responses, drug resistances followed by relapse invariably occurs.

In recent years, monoclonal antibodies have become an appealing approach in cancer therapy for several reasons. Antibodies can be highly specific for their targets and therefore off-target side effects are reduced compared to small molecule kinase inhibitors. Specific targeting of oncogenic RTKs by inhibitory monoclonal antibodies (mAb) may allow the resistance that frequently occurs in patients treated with kinase inhibitors to be surmounted. Indeed, several mAbs targeting RTKs have shown promising results as anticancer therapies. For example mAb against members of the ErbB family of RTKs were approved for clinical use including cetuximab and matuzumab (anti EGFR mAbs), as well as trastuzumab and pertuzumab (anti ErbB2 mAbs) (Cho et al. (2003) Nature 421: 756-760; Franklin et al.

(2004) Cancer Cell 5: 317-328; Li et al. (2005) Cancer Cell 7: 301-311; Schmiedel et al. (2008) Cancer Cell 13: 365-373). However, monoclonal antibodies targeting the KIT receptor tyrosine kinase that are therapeutically effective are still needed in the art.

Summary of the Invention

The present invention provides novel antibodies, and antigen-binding portions thereof, that bind the membrane proximal domain of the KIT receptor tyrosine kinase (RTK). Human anti-KIT antibodies were isolated from a naive, phage-display synthetic antibody library. Crystal structures of a Fab fragment (Fragment antigen binding) in complex with KIT membrane proximal domains D4 and D5 (KITm-s) revealed binding to D4 that overlapped significantly with an epitope required for homotypic interactions essential for SCF dependent KIT activation. Furthermore, information obtained from the structure of the antibodies in complex with ΚΓΓ was applied to guide the design of affinity maturation libraries, enabling isolation of human anti-KIT antibody variants with increased binding affinity for the antigen and, thus, increased biological efficacy. The novel anti-ΚΓΓ antibodies, and antigen-binding portions thereof, disclosed herein are capable of efficient inhibition of ΚΓΓ activity that leads to suppression of cell proliferation and provide a potential novel therapeutic approach for the treatment of diseases, such as cancers, associated with or caused by KIT or KIT mutants.

Accordingly, in one aspect, the invention provides an isolated human anti-KIT antibody, or antigen-binding portion thereof, wherein the antibody, or antigen-binding portion thereof, has one or more of the following biological characteristics: binds to human KIT with a K_a of at least 1 x 10⁶ M^"1 s^"1; binds to human KIT with a K_a of at least 1.4 x 10⁶ M^"1 s^"1; binds to human KIT with a K_a of at least 2.4 x 10⁶ M^"1 s^"1; binds to human KIT with a K_a of at least 1.4 x 10⁶ M^"1 s^"1; binds to the D4 domain of human KIT and blocks homotypic interactions between Arg381 and Glu 386; inhibits SCF-stimulated autophosphorylation of KIT; inhibits SCF-stimulated autophosphorylation of KIT at a concentration of about 5 nM to about 50 nM; inhibits SCF-stimulated cell proliferation; inhibits SCF-stimulated cell proliferation at a concentration of about 5 nM to about 50 nM; dissociates from human KIT with a Ka of 6.6 x 10^"4 s^"1 or less; dissociates from human KIT with a Ka of 3.2 x 10^"4 s^"1 or less; dissociates from human KIT with a Ka of 2.7 x 10^"4 s^"1 or less; dissociates from human KIT with a K_d of 2.7 x 10^~5 s^"1 or less; or dissociates from human KIT with a K_d of 1.5 x 10^~5 s^"1 or less.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, which binds to the same epitope as Fabl9, Fabl2I, or Fab79D.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, which binds to the same epitope as an antibody comprising the six CDRs of Fabl9, Fabl2I or Fab79D.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, which binds to amino acid residues Pro317-Asn320 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further binds to amino acid residues Glu 329-Asp332, Ile334, Glu336, Lys358, Glu360, Tyr362, Lys364, Glu366, Arg372, Glu376, His378, Thr380 and Arg381 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further binds to amino acid residues Phe316, Val325, Glu329-Asp332, Ee334, Glu336, Glu360, Tyr362-Lys364, Glu366, Arg372, Glu376, His378, Thr380 and Arg381 of human KIT.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a heavy chain complementary determining region (CDR) 3 which binds to amino acid residues Glu329, Val331, Asp332, Lys358, Glu360, Glu376, His378 and Thr380 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof,

further comprises a heavy chain CDR2 that binds to amino acid residues Pro317, Met318, Asn320, Ile334, Glu336, Lys364 and Arg372 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a heavy chain CDR1 that binds to amino acid residues Ile319, Asn320 and Glu329-Val331 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDR3 that binds to amino acid residues Tyr362, Glu366 and Arg381 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDR2 that binds to amino acid residues Tyr362, Glu366 and Arg381 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDR1 that does not bind human KIT.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a heavy chain complementary determining region (CDR) 3 which binds to amino acid residues Glu329-Asp332, Glu360, Glu376, His378 and Thr380 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a heavy chain CDR2 that binds amino acids Phe316-Asn320, Ile334, Glu336, Lys364 and Arg372 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a heavy chain CDRl that binds amino acids Ile319, Asn320, Val325, Asn330 and Glu360 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDR3 that binds amino acids Tyr362 and Glu376 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDR2 that binds amino acids Asn330, Thr380 and Arg381 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDRl that binds amino acids Glu360, Pro363, Lys364 and Glu366 of human KIT.

In another aspect, the invention provides an isolated anti-KIT antibody, or an antigen- binding portion thereof, wherein the human anti-KIT antibody comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:98. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:95 or SEQ ID NO:97. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a heavy chain CDRl domain comprising the amino acid sequence selected from the group consisting of of any one of SEQ ID NOs: 1, 13, 16, 19, 24, 63, 65, 94, 96, 101, 102, 104, 105, 106, 107, 108, 109, 110, 111 and 112. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9. In one embodiment, the antibody, or antigen- binding portion thereof, further comprises a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 100. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain CDRl domain comprising the amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 7, 29, 32, 35, 38, 41, 46, 49, 62, 64, 66, 99, 103, 113, 114, 115, 116, 117, 118, and 119.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising the six CDRs of Fabl9, Fabl2I or Fabs79A-H.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a heavy chain variable region of any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain variable region of any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60.

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a heavy chain variable region of any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58 and a light chain variable region of any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60.

In yet another aspect, the invention provides an isolated anti-KIT antibody, or antigen -binding portion thereof, comprising a VH CDRl having the amino acid sequence

ΧΎ 1 Γ2ΜΗ, wherein X 1¹ is one or zero amino acid residues, and X 2 is selected from the group consisting of S and M (SEQ ID NO:63); a VH CDR2 comprising the amino acid sequence of SIYPYSGYTYYADSVKG (SEQ ID NO:2); and a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3).

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a VL CDRl having the amino acid sequence

RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein X¹ is selected from the group consisting of S, I, R, P and N; X 2 is selected from the group consisting of V, R, Y G and F; X 3 is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R,

V and T, X⁵ is zero or one amino acid residues, X⁶ is zero or one amino acid residues, and X⁷ is zero or one amino acid residues (SEQ ID NO:64); a VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO:8); and a VL CDR3 having the amino acid sequence QQWAVHSLIT (SEQ ID NO:9).

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a VH CDRl having the amino acid sequence 1 2 wherein X 1 is one or zero amino acid residues, and X 2 is selected from the group consisting of S and M (SEQ ID NO:63); a VH CDR2 comprising the amino acid sequence of

SIYPYSGYTYYADSVKG (SEQ ID NO:2); a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3); a VL CDRl having the amino acid sequence RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein X¹ is selected from the group consisting of S, I, R, P and N; X 2 is selected from the group consisting of V, R, Y G and F; X 3 is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R,

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a VH CDRl having the amino acid sequence 1 2 wherein X¹ is an amino acid selected from the group consisting of no amino acid, S and V, and X² is selected from the group consisting of S and M (SEQ ID NO:65); a VH CDR2 comprising the amino acid sequence of SIYPYSGYTYYADSVKG (SEQ ID NO:2); and a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3).

RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein X¹ is selected from the group consisting of S, I, R, P and N; X 2 is selected from the group consisting of V, R, Y G and F; X 3 is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R, V and T, X⁵ is selected from the group consisting of no amino acid, L, R, N, S, V and R, X⁶ is selected from the group consisting of no amino acid, R, P, V and I, and X is selected from the group consisting of no amino acid, R, M or S (SEQ ID NO:66); a VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO:8); and a VL CDR3 having the amino acid sequence QQWAVHSLIT (SEQ ID NO:9).

In another aspect, the invention provides an isolated anti-KIT antibody, or antigen- binding portion thereof, comprising a VH CDRl having the amino acid sequence 1 2 wherein X¹ is an amino acid selected from the group consisting of no amino acid, S and V, and X² is selected from the group consisting of S and M (SEQ ID NO:65); a VH CDR2 comprising the amino acid sequence of SIYPYSGYTYYADSVKG (SEQ ID NO:2); a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3); a VL CDRl having the amino acid sequence RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein X¹ is selected from the group consisting of S, I, R, P and N; X is selected from the group consisting of V, R, Y G and F; X³ is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R, V and T, X⁵ is selected from the group consisting of no amino acid, L, R, N, S, V and R, X⁶ is selected from the group consisting of no amino acid, R, P, V and I, and X is selected from the group consisting of no amino acid, R, M or S (SEQ ID NO:66); a VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO:8); and a VL CDR3 having the amino acid sequence QQWAVHSLIT (SEQ ID NO:9). In one embodiment, the antibody or antigen-binding portion thereof, is a human antibody, a humanized antibody, a bispecific antibody, and a chimeric antibody. In another embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain constant region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions. In one embodiment, the heavy chain constant region is IgGl. In another embodiment, the antibody, or antigen-binding portion thereof, is a Fab fragment, a F(ab')₂ fragment or a single chain Fv fragment.

In one embodiment, the antibody, or antigen-binding portion thereof, binds to the D4 domain of human KIT and blocks homotypic interactions between Arg381 and Glu386. In another embodiment, the antibody, or antigen-binding portion thereof inhibits SCF- stimulated autophosphorylation of KIT. In another embodiment, the antibody, or antigen- binding portion thereof inhibits SCF-stimulated autophosphorylation of KIT at a

concentration of about 5 nM to about 50 nM. In one embodiment, the antibody, or antigen- binding portion thereof, inhibits SCF-stimulated cell proliferation. In another embodiment, the antibody, or antigen-binding portion thereof inhibits SCF-stimulated cell proliferation at a concentration of about 5 nM to about 50 nM.

In another embodiment, the antibody, or antigen-binding portion thereof, is conjugated to a different moiety. In one embodiment, the moiety is a toxin. In another embodiment, the moiety is an anti-cancer agent.

In another aspect, the invention provides a pharmaceutical composition comprising the antibody, or antigen-binding portion thereof, of the invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent.

In another aspect, the invention provides a hybridoma which produces the antibody, or antigen binding portion thereof, of the invention.

In another aspect, the invention provides methods of treating or preventing a KIT associated disease in a subject by administering to the subject an effective amount of the antibody, or antigen binding portion thereof, of the invention, thereby treating or preventing the disease in the subject. In one embodiment, the KIT associated disease is cancer, age- related macular degeneration (AMD), atherosclerosis, rheumatoid arthritis, diabetic retinopathy, or pain associated diseases. In another embodiment, the cancer is GIST, AML or SCLC. In another embodiment, the antibody, or antigen-binding portion thereof, is administered in combination with an additional therapeutic agent. In another aspect, the invention provides a method of inhibiting the phosphorylation of human KIT, the method comprising contacting human KIT with the antibody, or antigen- binding portion thereof, of the invention, thereby inhibiting the phosphorylation of human KIT.

In another aspect, the invention provides a method of inhibiting SCF-stimulated cell proliferation of a cell, the method comprising contacting the cell with the antibody, or antigen-binding portion thereof, of the invention, thereby inhibiting the SCF-stimulated cell proliferation of the cell.

In yet another aspect, the invention provides a method of inhibiting the interaction between D4 domains of human KIT monomers, the method comprising contacting the human KIT monomers with the antibody, or antigen-binding portion thereof, of the invention, thereby inhibiting the interaction between the D4 domains of the KIT monomers.

In another aspect, the invention provides a method of preventing the dimerization of human KIT monomers, the method comprising contacting a human KIT monomer with the antibody, or antigen-binding portion thereof, of the invention, thereby preventing the dimerization of human KIT monomers.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Figure 1 depicts the crystal structure of the Fabl9-KITD4-5 complex. (A) depicts schematic representation of wild type KIT as well as the KIT_D4_5 fragment, that was used in this manuscript. Glu381 and Arg386, residues important for KIT activation are marked on KIT_D4-5 fragment. D1-D5 are Ig-like domains 1 to 5 respectively, TM - transmembrane, JM - juxtamembrane, PTK - protein tyrosine kinase domain, KI - kinase insert. (B) Surface representation of Fab^-KIT^s complex. Left panel shows a view following 90° rotation along the shown axis. D4 and D5 of KIT are colored with orange and pink, and heavy and light chains of Fab 19 with blue and green, respectively. The regions of interactions between Fabl9 and KITD4-5 are visualized in Open book' format for D4 (C) and Fab (D). The buried surface area on the D4 side is shown in red and is in the same scale as in B (C). The interactions between Fab 19 and D4 are mediated by CDR loops and surrounding residues. The buried interface surface of the Fab 19 is color coded by CDR: HI - cyan, H2 - magenta, H3 - grey, L2- bright green, L3 - yellow (D). Fab 19 in panel D is enlarged compare to images shown in panels B and C.

Figure 2 depicts the Fabl9-KITD4-5 interface. Detailed view of the interactions between Fab 19 and KITD4-5 fragment; cartoon representation. Side chains mediating interactions are shown with sticks and hydrogen bonds with dashes. General color code is the same as in Figure 1; CDR HI (A) is in cyan, H2 (B) in magenta, H3 (C) in grey and L3 (D) in yellow.

Figure 3 depicts the D4-D4 homotypic interactions are occluded by Fab 19 binding. (A) Left panel - surface representation of Fabl9-KITD4-5 complex, the color code is the same as in Figure 1. Residues Arg381 and Glu386, that mediate D4-D4 homotypic interactions, are indicated by arrows. Right panel - close up view of the interactions between Arg381 and CDR L2 of Fabl9; cartoon representation. Side chains for Arg381, Glu386 (D4), Tyr49 (light chain) and main chain of Leu54 (light chain) are shown in sticks, hydrogen bonds are shown as dashes. (B) Left panel - surface representation of KIT-SCF 2:2 complex. D4 and D5 are colored as in Fab 19- KITm-5 complex in (A); Dl, D2 and D3 are in red and SCF is in brown. Right panel - cartoon representation of D4-D4 homotypic interactions, mediated by two salt bridges between Arg381 and Glu386. Arg381 and Glu386 side chains are shown with sticks, hydrogen bonds are shown as dashes. (C) and (D) the surface representation of KITm fragment. (C) Interface between KITm and Fab 19 and its overlap with D4-D4 homotypic interactions. The surface is colored according to whether it interacts with Fabl9 only (red), another D4 during ligand dependent dimerization (magenta), or both (yellow). (D)

Theoretical interface of KTN37 IgG. Residues, which are potentially involved into KTN37- D4 interactions (Figures 11 and 12) are colored with blue; residues involved into D4-D4 homotypic interactions are colored with magenta.

Figure 4 depicts the comparison of binding kinetics of anti-D4 Fabs to KITm-5 fragment. Surface Plasmon Resonance (SPR) analysis of binding of Fabl9, Fabl2I, Fab79D and Fab-KTN37 to immobilized ΚΠ Ε>4-5 fragment. Fabs in serial dilution (0.12 nM, 0.37 nM, l.lnM, 3.3 nM and 10 nM) were passed over Biosensor surface to which KITm-5 fragment was covalently coupled. (A) Association and dissociation kinetic curves are shown for each Fab. The data were fit into 1: 1 Langmuir interaction model (O'Shannessy et al. (1993) Anal Biochem 212: 457-468) (black lines) using Biacore T100 Evaluation software. In order to get reliable fitting, dissociation times were significantly increased for Fab79D and Fab 121 binding (Figure 12). (B) Values for association and dissociation constants were calculated using sensograms from (A).

Figure 5 depicts the comparison of Fabl9-KITD4-5 and Fab79D-KITD4-5 complex structures. (A) Structural alignment of VL, VH and D4 domains from Fabl9-KITD4-5 and Fab79D-KITD4-5 structures (ribbon representation). Fab79D complex is colored with: D4 - orange, VL - green and VH - cyan; Fabl9 complex: D4 - red, VL - yellow and VH - dark blue. CDRs LI are highlighted with magenta for Fab79D, and with black for Fabl9.B and C. Cartoon representation of contacts between CDR LI of Fabl9 (B) and Fab79D (C) and βϋ of D4. Side chains are shown as sticks and hydrogen bonds with dashes.

Figure 6 depicts KIT activation is inhibited by anti-D4 antibodies. (A) NIH 3T3 cells expressing wild type KIT (WT) were incubated for 5 hours with the indicated concentrations (upper panel) of Fabs or IgGs following stimulation with 25 ng/ml of SCF for 5 minutes at 37°C. Lysates of unstimulated or SCF- stimulated cells were subjected to

immunoprecipitation (IP) with anti-KIT antibodies followed by SDS-PAGE and

immunoblotting (IB) with either anti-KIT or anti-phosphotyrosine (pTyr) antibodies. (B) and (C) Ba/F3 cells expressing WT KIT were plated in 6 well plates and grown in the presence of 250 nM SCF and different concentrations (as indicated in the lower panel) of Fabs (B) or IgGs (C). Cell number were measured after 72 hour relative to 0 hour time points.

Figure 7 depicts proliferation of Ba/F3 cells expressing A502,Y503 duplication oncogenic KIT mutant is inhibited by anti-D4 antibodies. Ba/F3 cells expressing the dupA502,Y503 KIT mutant were plated in 6 well plates. Cells were grown in the presence of different concentrations of Fabs (A) or IgGs (B), in the absence of IL-3 or SCF (as indicated). Folds increase of cell number was measured after 72 hours.

Figure 8 depicts isolation of Fab 19 from a naive synthetic antibody library. (A) CDR sequences of a naive synthetic Fab library and Fabl9. (B) Flow cytometric analysis of purified Fab 19 against CHO cells overexpressing KIT. Percent staining is a quantification of the histogram below and indicates the proportion of the population staining positive with Fab 19 (red trace) above background controls with secondary antibody alone (black trace). Figure 9 depicts a diagram of amino acids mediating the interaction between Fab 19 and D4 of KIT. Diagrams were generated using the PDBsum server (Laskowski (2007) Bioinformatics 23: 1824-1827). Amino acids are colored according to their chemical properties; positive in blue, negative in red, neutral in green, aliphatic in gray and aromatic in purple. Interactions are depicted in blue solid and orange striped lines for hydrogen bonds and non-bonded contacts, respectively. For non-bonded contacts, the thickness of the lines corresponds to the number of contacts.

Figure 10 depicts isolation and characterization of affinity mature anti KIT D4 Fab. (A) CDR-H1 sequences of isolated affinity matured variants. (B) Multipoint ELISA to compare affinities of variants using serial dilutions of normalized phage and purified bound antigen. (C) Fluid phase competitive ELISA using a combination of phage and purified antigen in solution at specified concentrations that competes with constant concentrations of immobilized antigen to estimate binding affinity of affinity matured variants to KIT D4. (D) CDR-L1 and HI sequences of Fabs isolated in the second generation of affinity maturation.

Figure 11 depicts superposition of CDR HI of Fabl9 and Fab79D. Cartoon representation of Fabl9-KITD4-5 and Fab79D-KITD4-5 structures with side chains are shown with sticks and variable regions of heavy chains of Fab 19 and Fab79D were aligned. Heavy chain of Fab 19 is colored with cyan; heavy chain of Fab79D colored with blue; KIT D4 colored with orange. (A) Interaction of Ser/Val31^H with KIT D4 (residues I\e3l9^m,

Van323^D4 and Val331^D4). (B) Substitution of Ser33^H (Fabl9) to Met (Fab79D) increases hydrophobic interactions with Tyr51 stabilizing conformation of CDR HI loop.

Figure 12 depicts Surface Plasmon Resonance (SPR) analysis of Fab79D. SPR analysis of binding of Fab79D to immobilized KITm-5 fragment. The 600 sec dissociation time that were initially used, did not yield reliable fit. In order to obtain a reliable signal to noise ratio a longer dissociation time (7200 sec) was used. Both experiments were merged together and were fit into 1: 1 Langmuir interaction model (O'Shannessy et al. (1993) Anal Biochem 212: 457-468) (black lines) using Biacore T100 Evaluation software. Purple and cyan lines represent long dissociation experiment at 10 nM and 3.3 nM concentration of Fab79D, respectively.

Figure 13 depicts diagram of amino acids mediating the interaction between Fab79D and D4 of KIT. Diagrams were generated using the PDBsum server (Laskowski (2007) Bioinformatics 23: 1824-1827). Amino acids are colored according to their chemical properties; positive in blue, negative in red, neutral in green, aliphatic in gray and aromatic in purple. Interactions are depicted in blue solid and orange striped lines for hydrogen bonds and non-bonded contacts, respectively. For non-bonded contacts, the thickness of the lines corresponds to the number of contacts.

Figure 14 depicts electron density maps for Fabl9-KITD4-5 complex. Stereo views of electron density maps. Protein model is colored by chains: light chain of Fabl9 - yellow, heavy chain - magenta and KITm-5 - blue. (A) and (B) 2Eobs-Ecaic electron density map represented with blue (contour level 1.0) and gray (contour level 2.0) mesh and obs- caic is represented with red and green mesh. (C) - simulated annealing OMIT map (blue mesh).

Figure 15 depicts electron density map for Fab79D-KITD4-5 complex. Stereo views of electron density maps. Protein model is colored by chains: light chain of Fab79D - yellow, heavy chain - magenta and KITm-5 - blue. (A) and (B) 2Eobs-Ecaic electron density map represented with blue (contour level 1.0) and gray (contour level 2.0) mesh and obs- caic is represented with red and green mesh. (C) - simulated annealing OMIT map (blue mesh).

Figure 16 depicts anisotropy analysis of Fab 19-KITD4-5 processed data. Anisotropy analysis was done using the Diffraction Anisotropy Server at UCLA (Strong et al. (2006) Proc Natl Acad Sci USA 103: 8060-8065). The data for Fabl9-KITm-5 complex has

STRONG anisotropy based on the spread of values of the three principle components = 25.14 A².

Figure 17 depicts anisotropy analysis of Fab79D-KITD4-5 processed data. Anisotropy analysis was done using the Diffraction Anisotropy Server at UCLA (Strong et al. (2006) Proc Natl Acad Sci USA 103: 8060-8065). The data for Fab79D-KITm-5 complex has STRONG anisotropy based on the spread of values of the three principle components = 52.18 A².

Detailed Description of the Invention

The present invention provides antibodies, or antigen-binding portions thereof, that bind to the membrane proximal region, e.g., the D4 domain, of human KIT (also known as the SCF receptor), and methods of use thereof. The antibodies disclosed herein are capable of efficient inhibition of KIT activity that leads to suppression of cell proliferation and provide a novel therapeutic approach for the treatment of diseases, such as cancers, associated with or caused by wild-type KIT or KIT mutants. The antibodies, and antigen- binding portions thereof, of the invention may block the homotypic interaction of the D4 domains of two KIT monomers, thereby locking the ectodomain of human KIT in a monomeric state. In another embodiment of the invention, the antibodies, or antigen-binding portions thereof, of the invention allow the ectodomain of KIT to dimerize but affect the positioning, orientation and/or distance between the Ig-like domains of two KIT monomers (e.g., the D4-D4 or D5-D5 domains of human KIT), thereby inhibiting the activity of KIT. In other words, the antibodies of the invention may allow ligand induced dimerization of the KIT ectodomains, but affect the positioning of the two ectodomains at the cell surface interface or alter or prevent conformational changes in ΚΓΓ, thereby inhibiting the activity of KIT (e.g., inhibiting receptor internalization and/or inhibiting tyrosine autophosphorylation of the receptor and/or inhibiting the ability of KIT to activate a downstream signaling pathway).

The present invention is based, at least in part, on the identification of novel human anti-KIT antibodies and the deciphering of the crystal structures of the D4-D5 domains of KIT bound to anti-KIT Fab fragments. The deciphering of these crystal structures has allowed for the identification of epitopes, e.g., conformational epitopes, which the antibodies of the invention may bind, and has also allowed for structure-based affinity maturation to produce improved anti-KIT antibodies, and antigen-binding portions thereof.

In order that the present invention may be more readily understood, certain terms are first defined.

The terms "receptor tyrosine kinase" and "RTK" are used interchangeably herein to refer to the well-known family of membrane receptors that phosphorylate tyrosine residues. Many play significant roles in development or cell division. Receptor tyrosine kinases possess an extracellular ligand binding domain, a transmembrane domain and an intracellular catalytic domain. The extracellular domain binds cytokines, growth factors or other ligands and is generally comprised of one or more identifiable structural motifs, including cysteine- rich regions, fibronectin Ill-like domains, immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like domains, Factor VIITlike domains, glycine-rich regions, leucine-rich regions, acidic regions and discoidin-like domains. Activation of the

intracellular kinase domain is achieved by ligand binding to the extracellular domain, which induces dimerization of the receptors. A receptor activated in this way is able to

autophosphorylate tyrosine residues outside the catalytic domain, facilitating stabilization of the active receptor conformation. The phosphorylated residues also serve as binding sites for proteins which will then transduce signals within the cell. Examples of RTKs include, but are not limited to, KIT (also known as Stem Cell Factor receptor or SCF receptor), fibroblast growth factor (FGF) receptors, hepatocyte growth factor (HGF) receptors, insulin receptor, insulin-like growth factor- 1 (IGF-1) receptor, nerve growth factor (NGF) receptor, vascular endothelial growth factor (VEGF) receptor, PDGF-receptor-a, PDGF-receptor-β, CSF-1- receptor (also known as M-CSF-receptor or Fms), and the Flt3-receptor (also known as Flk2).

As used herein the term "type III family of receptor tyrosine kinases" or "type III RTKs" is intended to include receptor tyrosine kinases which typically contain five immunoglobulin like domains, or Ig-like domains, in their ectodomains. Examples of type III RTKs include, but are not limited to PDGF receptors, the M-CSF receptor, the FGF receptor, the Flt3-receptor (also known as Flk2) and the KIT receptor. In a preferred embodiment of the invention, the type III RTK is KIT (also known in the art as the SCF receptor). KIT, like other type III RTKs is composed of a glycosylated extracellular ligand binding domain (ectodomain) that is connected to a cytoplasmic region by means of a single transmembrane (TM) domain (reviewed in Schlessinger (2000) Cell 103: 211-225). Another hallmark of the type III RTKs, e.g., KIT, is a cytoplasmic protein tyrosine kinase (PTK) domain with a large kinase-insert region. At least two splice isoforms of the KIT receptor are known to exist, the shorter making use of an in-frame splice site. All isoforms of KIT, and the other above described RTKs, are encompassed by the present invention.

The terms "Kit", "KIT" and "KIT receptor", as used herein, include the type III transmembrane receptor tryosine kinase (RTK) that plays crucial roles in mediating diverse cellular processes including cell differentiation, proliferation and cell survival, among other activities, upon binding by any KIT ligand, e.g., Stem Cell Factor (SCF) (reviewed in

Schlessinger (2000) Cell 103: 211-225). KIT is also known as the SCF receptor. Like other members of the type III subfamily of RTKs, KIT is composed of an extracellular domain that includes five Ig-like domains (designated D1-D5), a single transmembrane domain, a juxtamembrane region , a tyrosine kinase domain split by a kinase insert and a C-terminal tail. In one embodiment of the invention, the KIT is human KIT. The term "KIT" is also intended to include recombinant human KIT (rh KIT), which can be prepared by standard recombinant expression methods.

The Genbank reference sequence for the human KIT mRNA NM_000222.2 (encoding the protein NP_000213.1) is as follows:

MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCT DPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAK LFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKS

VKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVT

CTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFM

CYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQ

WIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIA

FNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQT

LNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPH

TLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLP

YDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTE

REALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFIC

SKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYI

ERDVTKITMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRIT

KICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELF

SLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQI

VQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

(SEQ ID NO: 92).

Similarly, the Genbank reference sequence for variant 2 of the KIT mRNA

NM_001093772.1 (encoding protein NP_001087241.1) is as follows:

MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCT

DPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAK

LFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKS

VKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVT

CTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFM

CYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQ

WIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIA

FNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQT

LNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTP

LLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHK

WEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREAL

MSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQE

DHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERD

VTKITMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKIC

DFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLG SSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQL IEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV (SEQ ID NO: 93).

As used herein, an "Ig-like domain" of KIT is intended to include the domains well known in the art to be present in the ectodomain of KIT. In the ectodomain of the family of type III receptor tyrosine kinases (type III RTKs), e.g., KIT, there are five such domains, known as Dl, D2, D3, D4 and D5. The Dl, D2 and D3 domains of type III RTKs are responsible for binding the ligand of the RTK (reviewed in Ullrich and Schlessinger (1990) Cell 61: 203-212). Thus, in one embodiment of the invention the term "Ig-like domain" is not intended to include a domain of KIT which is responsible for ligand binding. In a preferred embodiment of the invention, the Ig-like domain is a D4 and/or a D5 domain of KIT.

The term "ectodomain" of KIT is well known in the art and refers to the extracellular part of the KIT receptor, i.e., the part of the KIT receptor that is outside of the plasma membrane.

The term "a membrane proximal region" of the ectodomain of KIT refers to an extracellular part of the KIT receptor which is in proximity to the plasma membrane and which, preferably, is not directly responsible for the binding of a ligand to the KIT receptor. Examples of membrane proximal regions include, but are not limited to, the D4 domain of KIT, the D5 domain of KIT, the D3-D4 hinge region of KIT, and the D4-D5 hinge region of KIT.

The term "homotypic interaction" as used herein, refers to the interaction between two identical membrane proximal regions from two monomeric receptors.

The term "heterotypic interaction" as used herein, refers to the interaction between two different membrane proximal regions from two monomeric receptors. A heterotypic interaction may be the result of dimerization of two different types of monomeric receptors or the result of dimerization of a wild type and a mutant form of the same monomeric receptor. For example, it is well known in the art that a cancer patient may carry a wild type allele and a mutant allele for a certain receptor.

The term "monomeric state" as used herein, refers to the state of a RTK, such as KIT, wherein the RTK molecule is composed of a single polypeptide chain which is not associated with a second RTK polypeptide of the same or different type. RTK dimerization leads to autophosphorylation and receptor activation. Thus, a RTK in a monomeric state is in an inactive state. A monomelic state is also a state wherein the D4 or D5 domain of a single RTK, such as KIT, is not associated with the D4 or D5 domain, respectively, of a second, RTK, such as KIT.

The phrase "locks the ectodomain of KIT in an inactive state" refers to the ability of an antibody, or antigen-binding portion thereof, of the invention to inhibit the activity of KIT. In other words, this phrase includes the ability of an antibody of the invention to shift the equilibrium towards formation of an inactive or inhibited ΚΓΓ receptor configuration. For example, an antibody, or antigen-binding portion thereof, of the invention may inhibit the activity of KIT by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% as compared to the activity of the KIT receptor in the absence of the antibody, or antigen-binding portion thereof, .

The term "inactive state," as used herein, refers to the state of a KIT receptor wherein the KIT receptor molecule is unable to activate downstream signaling. An inactive state may be a state wherein the ectodomain of the KIT receptor is allowed to dimerize but the positioning, orientation, conformation, and/or distance between the Ig-like domains of the two monomers (e.g., the D4-D4 or D5-D5 domains of the KIT receptor), is altered such that the activity of the KIT receptor is inhibited (e.g., receptor internalization is inhibited and/or tyrosine autophosphorylation of the receptor is inhibited and/or the ability of the receptor to activate a downstream signaling pathway is inhibited). An inactive state also includes a monomeric state as described above. An inactive state may also be a state in which the ectodomain of the KIT receptor is bound to a receptor ligand and is dimerized, but has not yet undergone the conformational change that allows for the activation of the receptor. The term "inactive state" includes a state in which an antibody, or antigen-binding portion thereof, of the invention may reduce or inhibit the activity of the KIT receptor by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% as compared to the activity of the receptor in the absence of the antibody, or antigen- binding portion thereof. Any of the functional assays described herein may be used to determine the ability of an antibody, or antigen-binding portion thereof, of the invention to inhibit the activity of the KIT receptor.

As used herein, the terms "conformational epitope" or "non-linear epitope" or "discontinuous epitope" are used interchangeably to refer to an epitope which is composed of at least two amino acids which are not consecutive amino acids in a single protein chain. For example, a conformational epitope may be comprised of two or more amino acids which are separated by a stretch of intervening amino acids but which are close enough to be recognized by an antibody, or antigen-binding portion thereof, of the invention as a single epitope. As a further example, amino acids which are separated by intervening amino acids on a single protein chain, or amino acids which exist on separate protein chains, may be brought into proximity due to the conformational shape of a protein structure or complex to become a conformational epitope which may be bound by an antibody, or antigen-binding portion thereof, of the invention. Particular discontinuous and conformation epitopes are described herein (see, for example, the Examples below).

It will be appreciated by one of skill in the art that, in general, a linear epitope bound by an antibody, or antigen-binding portion thereof, of the invention may or may not be dependent on the secondary, tertiary, or quaternary structure of the KIT receptor. For example, in some embodiments, an antibody, or antigen-binding portion thereof, of the invention may bind to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure. In other embodiments, an antibody, or antigen- binding portion thereof, of the invention may not recognize the individual amino acid residues making up the epitope, and may require a particular conformation (bend, twist, turn or fold) in order to recognize and bind the epitope.

The term "polypeptide" as used herein, refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may be monomeric or polymeric.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

The term "recovering" as used herein, refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.

"Biological activity" as used herein, refers to all inherent biological properties of the KIT receptor. Biological properties of KIT include but are not limited to binding of SCF; other examples include cell differentiation, proliferation and cell survival.

The term "antibody", as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains. The term "antibody", as used herein, also refers to any antigen-binding portion, mutant, variant, or derivative of an immunoglobulin molecule, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art and nonlimiting embodiments of which are discussed herein.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., KIT, such as the D4 domain of KIT). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi- specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546, Winter et al, PCT publication WO 90/05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354- 5).

The term "antibody construct" as used herein refers to a polypeptide comprising one or more of the antigen binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

An antibody or antigen-binding portion thereof may be part of a larger

immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such

immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93- 101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.

Immunol. 31: 1047-1058). Antibody portions, such as Fab and F(ab')₂ fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities {e.g., an isolated antibody that specifically binds the D4 domain of KIT is substantially free of antibodies that specifically bind antigens other than the D4 domain of KIT). An isolated antibody that specifically binds the D4 domain of human KIT may, however, have cross-reactivity to other antigens, such as the D4 domain of KIT molecules from other species. Alternatively, an isolated antibody, or antigen-binding portion thereof, may not cross-react with the D4 domain of KIT molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences {e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. In another embodiment, the human monoclonal antibodies are produced by phage display technologies as described, for example, in the Examples section below.

The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29: 128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human

immunoglobulin genes (see e.g., U.S. Patent No. 6,713,610; Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.

The term "chimeric antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

The term "CDR-grafted antibody" refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.

The term "humanized antibody" refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Such antibodies were generated by obtaining murine anti-KIT monoclonal antibodies using traditional hybridoma technology followed by humanization using in vitro genetic engineering.

The term "antibody mimetic" or "antibody mimic" is intended to refer to molecules capable of mimicking an antibody's ability to bind an antigen, but which are not limited to native antibody structures. Examples of such antibody mimetics include, but are not limited to, Adnectins (i.e., fibronectin based binding molecules), Affibodies, DARPins, Anticalins, Avimers, and Versabodies all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms. The embodiments of the instant invention, as they are directed to antibodies, or antigen binding portions thereof, also apply to the antibody mimetics described above.

As used herein, "isotype" refers to an antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

The terms "Kabat numbering", "Kabat definitions and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

As used herein, the terms "acceptor" and "acceptor antibody" refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). In yet another embodiment, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In accordance with this embodiment, an acceptor may contain at least 1, at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and/or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).

As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et ah, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al, Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 and L3 or HI, H2 and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not

significantly impact antigen binding.

The international ImMunoGeneTics database ("IMGT") numbering system has been defined to compare the variable regions and domains of any antigen receptor, including immunoglobulins (Lefranc et al., Dev. Comp. Immunol. 27: 55-77 (2003)). The system combines the definition of the framework and CDR, structural data, and the characterization of hypervariable regions. The IMGT numbering system relies on the high conservation of structure within the variable region, whereby all conserved amino acids from frameworks always occupy the same position. For example cysteine 23, tryptophan 41, hydrophobic amino acid 89, cysteine 104, and phenylanaline or tryptophan 118 always occupy the same position, regardless of the immunoglobulin variable sequence that they come from. The IMGT numbering system provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1-26; FR2-IMGT:positions 39-55; FR3-IMGT: positions 66-104; FR4-IMGT: positions 118-128) and of the CDRs (CDR1-IMGT: positions 27-38; CDR2- IMGT: positions 56-65; CDR3-IMGT: positions 105-116). Shorter CDRs may be

represented by gaps. The IMGT numbering system allows for the standardized analysis and graphical representation of sequences designated IMGT Colliers de Perles (available on the IMGT databases website).

The methods used herein may utilize CDRs defined according to any of the above systems, although preferred embodiments use Kabat, Chothia, or IMGT defined CDRs. In one embodiment, the instant invention may utilize CDRs defined by the Kabat system. In another embodiment, the instant invention may utilize CDRs defined by the Chothia system. In another embodiment, the instant invention may utilize CDRs defined by the IMGT system.

As used herein, the term "canonical" residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al, J. Mol. Biol. 227:799 (1992), both are incorporated herein by reference). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.

As used herein, the terms "donor" and "donor antibody" refer to an antibody providing one or more CDRs. In a preferred embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non- human antibody providing one or more CDRs.

As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub- regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in the art.

As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular immunoglobulin. (See, e.g., Shapiro et ah, Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al, Adv Exp Med. Biol. 484: 13-30 (2001)). One of the advantages of germline antibody genes stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species.

As used herein, the term "key" residues refer to certain residues within the variable region that have more impact on the binding specificity and/or affinity of an antibody, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (can be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term "humanized antibody" is an antibody or a variant, derivative, analog or fragment thereof which binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')₂, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or humanized heavy chain.

The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. Such mutations, however, will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

As used herein, "Vernier" zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.

The term "multivalent binding protein" is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is preferably engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term "multispecific binding protein" refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins as used herein, are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e. capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site.

As used herein, the term "neutralizing" refers to neutralization of biological activity of a receptor when a binding protein specifically binds the receptor. Preferably a neutralizing binding protein is a neutralizing antibody whose binding to KIT and/or a mutant KIT protein results in inhibition of a biological activity of KIT and/or the mutant KIT protein. Preferably the neutralizing binding protein binds KIT and/or a mutant KIT protein and reduces a biologically activity of KIT and/or a mutant KIT protein by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more. Inhibition of a biological activity of KIT and/or a mutant KIT protein by a neutralizing binding protein can be assessed by measuring one or more indicators of KIT and/or mutant KIT biological activity well known in the art. In one embodiment, inhibition of KIT receptor autophosphorylation can be measured. In another embodiment, inhibition of KIT mediated cell proliferation can be measured.

The term "activity" includes activities such as the binding specificity/affinity of an antibody for an antigen, for example, an anti-KIT antibody that binds to a KIT antigen and/or the neutralizing potency of an antibody, for example, an anti-KIT antibody whose binding to KIT inhibits the biological activity of KIT, e.g., inhibits ΚΓΓ receptor autophosphorylation and KIT mediated cell proliferation.

The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

The term "surface plasmon resonance", as used herein, refers to an optical

phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the

BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The terms "specific binding" or "specifically binding", as used herein, in reference to the interaction of an antibody with another moiety, e.g., the KIT receptor or a portion thereof, such as the D4 domain, mean an interaction that is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the moiety, e.g., the KIT receptor or a portion thereof, such as the D4 domain. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins, generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

As used herein, an antibody that "binds" or "specifically binds" to an antigen, e.g., KIT or a fragment of KIT, such as the D4 domain of KIT, is intended to refern to an antibody, or antigen-binding portion thereof, that specifically binds to the antigen. The term "K_on" (also "Kon", "kon"), as used herein, is intended to refer to the on rate constant for association of a binding protein of the invention (e.g., an antibody of the invention) to an antigen to form an association complex, e.g., antibody/antigen complex, as is known in the art. The "K_on" also is known by the terms "association rate constant", or "ka", as used interchangeably herein. This value indicates the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen as is shown by the equation below:

Antibody ("Ab") + Antigen ("Ag")→Ab-Ag.

The term "K₀ff" (also "Koff", "koff"), as used herein, is intended to refer to the off rate constant for dissociation, or "dissociation rate constant", of a binding protein of the invention (e.g., an antibody of the invention) from an association complex (e.g., an antibody/antigen complex) as is known in the art. This value indicates the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation below:

Ab + Ag^ Ab-Ag.

The term "K_D" (also "K^"), as used herein, is intended to refer to the "equilibrium dissociation constant", and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). The association rate constant (Kon), the dissociation rate constant (Koff), and the equilibrium dissociation constant (K are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.

The term "labeled binding protein" as used herein, refers to a protein with a label incorporated that provides for the identification of the binding protein. Preferably, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ^mIn, ¹²⁵I, ¹³¹I,

ITT 166 153

Lu, Ho, or Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase);

chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates. Representative examples of labels commonly employed for immunoassays include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectably labeled but may become detectable upon reaction with yet another moiety. Use of the term "detectably labeled" is intended to encompass the latter type of detectable labeling.

The term "antibody conjugate" refers to a binding protein, such as an antibody, linked, e.g., chemically linked, to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

Preferably the therapeutic or cytotoxic agents include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

The terms "crystal", and "crystallized" as used herein, refer to a protein, including an antibody, or antigen-binding portion thereof, that exists in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three- dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the "unit cell" of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1- 16, Oxford University Press, New York, N.Y., (1999)."

The term "polynucleotide" as referred to herein, means a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA but preferably is double- stranded DNA.

The term "isolated polynucleotide" as used herein shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature; is operably linked to a polynucleotide to which it is not linked in nature; or does not occur in nature as part of a larger sequence.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Protein constructs of the present invention may be expressed, and purified using expression vectors and host cells known in the art, including expression cassettes, vectors, recombinant host cells and methods for the recombinant expression and proteolytic processing of recombinant polyproteins and pre-proteins from a single open reading frame (e.g., WO 2007/014162, the entire contents of which are incorporated herein by reference).

"Transformation", as defined herein, refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time. The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Preferably host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Preferred eukaryotic cells include protist, fungal, plant and animal cells. Most preferably host cells include but are not limited to the prokaryotic cell line E. Coli; mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

The terms "regulate" and "modulate" as used interchangeably, and refer to a change or an alteration in the activity of a molecule of interest (e.g., the biological activity of KIT). Modulation may be an increase or a decrease in the magnitude of a certain activity or function of the molecule of interest. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.

Correspondingly, the term "modulator," as used herein, is a compound capable of changing or altering an activity or function of a molecule of interest (e.g., the biological activity of KIT). For example, a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule. Modulators encompassed by the present invention include the anti-KIT antibodies described herein, as well as fragments, conjugates, derivatives or variants of these antibodies.

The term "agonist", as used herein, refers to a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the agonist.

The term "antagonist" or "inhibitor", as used herein, refer to a modulator that, when contacted with a molecule of interest causes a decrease in the magnitude of a certain activity or function of the molecule compared to the magnitude of the activity or function observed in the absence of the antagonist. Particular antagonists of interest include those that block or modulate the biological activity, e.g., tyrosine kinase activity, of the KIT receptor.

The phrase "inhibit binding to the receptor" refers to the ability of an antibody, or antigen-binding portion thereof, to prevent the binding of SCF or another ligand to the KIT receptor. Such inhibition of binding to the receptor would result in diminishing or abolishing the biological activity mediated by binding of SCF or another ligand to the KIT receptor.

As used herein, the term "effective amount" refers to the amount of a therapy which is sufficient to reduce or ameliorate the severity and/or duration of a disorder or one or more symptoms thereof, prevent the advancement of a disorder, cause regression of a disorder, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).

The term "sample", as used herein, is used in its broadest sense. A "biological sample", as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.

The term "dosing" or "dose" or "dosage", as used herein, refers to the administration of a substance (e.g., an anti-KIT antibody, or antigen-binding portion thereof) to achieve a therapeutic objective (e.g., the treatment or amelioration of a symptom of cancer).

The term "combination" as in the phrase "a first agent in combination with a second agent" includes co-administration of a first agent and a second agent, which for example may be dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent. The present invention, therefore, includes methods of combination therapeutic treatment and combination pharmaceutical compositions. The term "combination therapy", as used herein, refers to the administration of two or more therapeutic substances, e.g., an anti-KIT antibody, or antigen-binding portion thereof, and another drug. The other drug(s) may be administered concomitant with, prior to, or following the administration of the anti-KIT antibody.

The term "concomitant" as in the phrase "concomitant therapeutic treatment" includes administering an agent in the presence of a second agent. A concomitant therapeutic treatment method includes methods in which the first, second, third, or additional agents are co-administered. A concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered. A concomitant therapeutic treatment method may be executed step-wise by different actors. For example, one actor may administer to a subject a first agent and a second actor may to administer to the subject a second agent, and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and additional agents) are after administration in the presence of the second agent (and additional agents). The actor and the subject may be the same entity (e.g., human).

Various aspects of the invention are described in further detail in the following subsections:

I. Anti-KIT Antibodies, and Antigen-Binding Portions Thereof

This invention provides anti-KIT antibodies, or antigen-binding portions thereof. In one embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, are human antibodies, or antigen-binding portions thereof. Exemplary antibodies are provided herein. The features of such exemplary antibodies are set forth in the Sequence Listing, tables, and Examples.

1. Variable Regions

The antigen-binding portion of an antibody comprises one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human KIT, or a portion thereof, such as the D4 domain of KIT). It has been shown that the antigen -binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, the light chain variable domain (VL) and the heavy chain variable domain (VH), are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for function in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

The anti-KIT antibodies of the present invention comprise at least one antigen binding domain. See Table 1 and the Sequence Listing for a representation of VH and VL sequences encompassed in the present invention which can be present in any combination to form an anti-KIT antibody of the present invention. In one embodiment, the VH is selected from any of the VH domains shown in SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 and 58.

In another embodiment, the VL is selected from any of the VL domains shown in SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 and 60.

In certain embodiments, the anti-KIT antibodies are human or chimeric antibodies or antibody fragments. In certain embodiments, the anti-KIT antibodies are human antibodies or antibody fragments. In other embodiments, the anti-KIT antibodies are human or chimeric antibodies or antibody fragments that bind to human KIT and inhibit KIT activity. In further embodiments, the anti-KIT antibodies are human or chimeric antibodies or antibody fragments that bind to human KIT and block homotypic interactions between Arg381 and Glu386 of human KIT, inhibit SCF-stimulated autophosphorylation of KIT, and/or inhibit SCF-stimulated cell proliferation.

In certain embodiments, the anti-KIT antibodies comprise a VH and/or VL domain that has a given percent identify to at least one of the VH and/or VL sequences disclosed in Table 1 and the Sequence Listing. As used herein, the term "percent (%) sequence identity", also including "homology" is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The anti-KIT antibodies of the invention may comprise, or have, a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, identity to the amino acid sequence of any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 and 58.

The anti-KIT antibodies of the invention comprising a VH amino acid sequence with a given percent identify to any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 and 58 may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

The anti-KIT antibodies of the invention may comprise a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 and 58. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

The anti-KIT antibodies of the invention may comprise, or have, a VL domain amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 and 60.

The anti-KIT antibodies of the invention comprising a VL amino acid sequence with a given percent identify to any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 and 60 may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

The anti-KIT antibodies of the invention may comprise, or have, a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 and 60. In certain embodiments, the substitutions are conservative amino acid substitutions. These anti- KIT antibodies have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 and 58, and a VL amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of the amino acid sequences of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 and 60. These anti-KIT antibodies may have at least two more more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological

characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 and 58, and a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 and 60. These anti-KIT antibodies may have at least two more more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 4. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein. In a further embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 4. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies of the invention comprise, or have, a VL amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 10. These anti- KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies of the invention comprise, or have, a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue

substitutions have been made relative to SEQ ID NO: 10. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 4, and a VL amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 10. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 4, and a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 10. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more 100% identity to the amino acid sequence of SEQ ID NO: 53. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a further embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been mdae relative to SEQ ID NO: 53. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VL amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 55. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 55. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies of the invention comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 53, and a VL amino acid sequence comprising, or having, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 55. These anti-KIT antibodies may have any two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 53, and a VL amino acid sequence comprising, or having, an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 55. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies of the invention comprise, or have, a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 20. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a further embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 20. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5,

6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies of the invention comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 20, and a VL amino acid sequence comprising, or having, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 10. These anti-KIT antibodies may have any two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 20, and a VL amino acid sequence comprising, or having, an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 10. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies of the invention comprise, or have, a VL amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 39. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6,

7, 8, 9, 10 or more) of the biological characteristics described herein

In certain embodiments, the anti-KIT antibodies comprise, or have, a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 39. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein . In a specific embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 20, and a VL amino acid sequence comprising, or having, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 39. These anti-KIT antibodies may have any two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 20, and a VL amino acid sequence comprising, or having, an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 39. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more 100% identity to the amino acid sequence of SEQ ID NO: 58. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a further embodiment, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 58. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies comprise, or have, a VL amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 60. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VL amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 60. The substitutions may be conservative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In a specific embodiment, the anti-KIT antibodies of the invention comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 58, and a VL amino acid sequence comprising, or having, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 60. These anti-KIT antibodies may have any two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 58, and a VL amino acid sequence comprising, or having, having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to SEQ ID NO: 60. The substitutions may be conserviative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In other embodiments, the anti-KIT antibodies of the invention, comprise, or have, a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a variable heavy (VH) amino acid sequence set forth in Table 2, and a VL amino acid sequence, comprising, or having, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a variable light (VL) amino acid sequence set forth in Table 2. These anti-KIT antibodies may have any two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In other embodiments, the anti-KIT antibodies comprise, or have, a VH amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to a variable heavy (VH) amino acid sequence set forth in Table 2, and a VL amino acid sequence comprising, or having, having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue substitutions have been made relative to a variable light (VL) amino acid sequence set forth in Table 2. The substitutions may be conserviative amino acid substitutions. These anti-KIT antibodies may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein. 2. Complementarity Determining Regions (CDRs)

Although the variable domain (VH and VL) comprises the antigen-binding region, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in segments called Complementarity Determining Regions (CDRs), both in the light chain (VL or VK) and the heavy chain (VH) variable domains. The more highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et ah, Supra). The three CDRs of the heavy chain are designated CDR-H1, CDR-H2, and CDR-H3, and the three CDRs of the light chain are designated CDR-Ll, CDR-L2, and CDR-L3. For the Kabat numbering system, CDR-H1 begins at approximately amino acid 31 {i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tyrosine residue. CDR-H2 begins at the fifteenth residue after the end of CDR- HI, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR- H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-Ll begins at approximately residue 24 {i.e., following a cysteine residue);

includes approximately 10-17 residues; and ends at the next tyrosine residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-Ll and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2; includes approximately 7-11 residues and ends at the sequence F-G-X-G, where X is any amino acid. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences).

In one embodiment, the instant invention provides CDR sequences with boundaries defined by the Kabat numbering system. In another embodiment, the invention provides CDR sequences with boundaries defined by the Chothia numbering system. In another embodiment, the invention provides CDR sequences with boundaries defined by the IMGT numbering system.

The anti-KIT antibodies of the invention comprise at least one antigen binding domain that comprises at least one complementarity determining region (CDR1, CDR2 and CDR3). In one embodiment, the anti-KIT antibodies comprise a VH that comprises at least one VH CDR (e.g., CDR-Hl, CDR-H2 or CDR-H3). In another embodiment, the anti-KIT antibodies comprise a VL that comprises at least one VL CDR (e.g., CDR-Ll, CDR-L2 or CDR-L3).

In certain embodiments, anti-KIT antibodies of the invention comprise a combination of any CDR-Hl sequence of Table 1, Table 2, Table 4, or the Sequence Listing; any CDR-H2 sequence of Table 1, Table 2, Table 4, or the Sequence Listing; any CDR-H3 sequence of Table 1, Table 2, Table 4 or the Sequence Listing; any CDR-Ll sequence of Table 1, Table 2, Table 4 or the Sequence Listing; any CDR-L2 sequence of Table 1, Table 2, Table 4, or the Sequence Listing; and any CDR-L3 sequence of Table 1, Table 2, Table 4, or the Sequence Listing, wherein the antibody binds human KIT. In certain embodiments, the antibody is an antibody fragment. In certain embodiments, the antibody is a human, humanized or chimeric antibody. In certain embodiments, such an antibody has at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In certain embodiments, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise, or have,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NOs: 1, 13, 16, 19, and 24,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 2 and/or

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NOs: 94, 101, 106, 108 and in,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 95 and/or

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NOs: 96, 102, 104, 105, 107, 109 and 112,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 97 and/or

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 98. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise, or have,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 1,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 13,

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein. The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise, or have,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 16,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 19,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 24,

The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise, or have, (a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 94,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 101,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 106,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 108, (b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 95 and/or

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 111,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 96,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 102,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 97 and/or (c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 98. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 104,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 105,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 107,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 109,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 112,

In another embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise, or have,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NOs: 7, 29, 32, 35, 38, 41, 62, 46 and 49,

(b) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and/or

(c) a VL CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein. In another embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise, or have,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NOs: 99, 103, 113, 114, 115, 116, 117, 118 and 119,

(b) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 100 and/or

(c) a VL CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 7,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 29,

The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise, or have, (a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 32,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 35,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 38,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 41, (b) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and/or

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 62,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 46,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 49,

(b) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and/or (c) a VL CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 99,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 103,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 113,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 114,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 115,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 116,

(c) a VL CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9. These anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein. The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise, or have,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 117,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 118,

(a) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 119,

The present invention contemplates antibodies, and antigen-binding portions thereof, comprising or having any combination of the foregoing VH and VL CDRs. For example, antibodies comprising: (a) a VH CDR1 having a sequence identical to SEQ ID NO: 1 or SEQ ID NO: 19,

(b) a VH CDR2 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 2, and

(c) a VH CDR3 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 3.

The present invention contemplates antibodies, and antigen-binding portions thereof, comprising or having any combination of the foregoing VH and VL CDRs. For example, antibodies comprising:

(a) a VH CDR1 having a sequence identical to SEQ ID NO: 94 or SEQ ID NO: 101,

(b) a VH CDR2 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 95, and

(a) a VH CDR1 having a sequence identical to SEQ ID NO: 96 or SEQ ID NO: 102,

(b) a VH CDR2 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 97, and

(c) a VH CDR3 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 98.

By way of further example, the presention invention further provides antibodies, or antigen-binding antibody fragments thereof, comprising:

(a) a VL CDR1 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 7 or SEQ ID NO:38,

(b) a VL CDR2 having an amino acid sequence identical to SEQ ID NO: 8, and

(c) a VL CDR3 having an amino acid sequence comprising two substitutions relative to SEQ ID NO: 9.

(a) a VL CDR1 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 99 or SEQ ID NO: 103,

(b) a VL CDR2 having an amino acid sequence identical to SEQ ID NO: 100, and (c) a VL CDR3 having an amino acid sequence comprising two substitutions relative to SEQ ID NO: 9.

All other combinations are similarly contemplated. In certain embodiments, the antibodies or antibody fragments are human antibodies that bind to human KIT and block homotypic interactions between Arg381 and Glu386 of the D4 domain of KIT, inhibit SCF- stimulated autophosphorylation of KIT, and/or inhibit SCF- stimulated cell proliferation.

The foregoing description of the VH and VL domains is intended to refer to all possible combinations. Thus, for example, description of a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid substitutions relative to, SEQ ID NO: 19 refers to any of the following embodiments:

(a) a VH CDRl having an amino acid sequence identical to SEQ ID NO: 19;

(b) a VH CDRl having an amino acid sequence comprising 1 amino acid residue substitution relative to SEQ ID NO: 19;

(c) a VH CDR 1 having an amino acid sequence comprising 2 amino acid residue substitutions relative to SEQ ID NO: 19;

(d) a VH CDRl having an amino acid sequence comprising 3 amino acid residue substitutions relative to SEQ ID NO: 19.

In certain embodiments, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise:

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 1,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 2,

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 7,

(e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and

(f) a VL CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9.

In certain embodiments, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise: (a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 94,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 95,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 7,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 97,

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 98,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 99,

(e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 100 and

The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise:

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 2, (c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3,

(f) a VL CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9.

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 38,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 7, (e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and

(f) a VL CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9. The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise:

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 29,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 32,

(a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 19, (b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 2,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 35,

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3, (d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 41,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 19,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 62,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 46,

(e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and (f) a VL CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9.

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 49,

The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise: (a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 101,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 95, (c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 41, (e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8 and

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 62,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 46,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 101,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 49,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 102,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 103,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 102, (b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 97,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 113,

(c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 98, (d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 114,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 115,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 116,

(e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 100 and (f) a VL CDR3 having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 9.

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 117,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 118,

The anti-KIT antibodies, or antigen-binding portions thereof, of the invention may comprise: (a) a VH CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 102,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 119,

(b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 97, (c) a VH CDR3 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 98,

(d) a VL CDR1 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 99, (e) a VL CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 100 and

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 108,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 109,

(d) a VL CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 99,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 110,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 111,

(a) a VH CDRl having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 112, (b) a VH CDR2 having an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 97,

The invention contemplates antibodies, and antigen-binding antibody fragments thereof, comprising or having any combination of the foregoing VH and VL CDRs. For example, antibodies comprising:

(a) a VH CDR1 having a sequence identical to SEQ ID NO: 19,

(b) a VH CDR2 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 2,

(c) a VH CDR3 having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 3,

(d) a VL CDR1 having an amino acid sequence comprising one substitution relative to SEQ ID NO: 7,

(e) a VL CDR2 having an amino acid sequence identical to SEQ ID NO: 8, and

(f) a VL CDR3 having an amino acid sequence comprising three substitutions relative to SEQ ID NO: 9.

All other combinations are similarly contemplated. In certain embodiments, the antibodies or antibody fragments are human antibodies that bind to human KIT and inhibit KIT activity, such as blocking homotypic interactions between Arg381 and Glu386 of the D4 domain of human KIT, inhibiting SCF- stimulated autophosphorylation of KIT, and/or inhibiting SCF-stimulated cell proliferation.

In a specific embodiment, the present invention provides an anti-KIT antibody, or antigen -binding fragment thereof, which comprises or has:

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 1;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(f) a VL CDR3 having the amino acid sequence of SEQ ID NO: 9. In one embodiment, these anti-KIT antibodies, or antigen-binding fragments thereof, have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In another embodiment, the present invention provides an anti-KIT antibody, or antigen-binding fragment thereof, which comprises or has:

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 38;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 13;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 16;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 24;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 29;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 32;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 35;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 41 ;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 62;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 46;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 19;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 49;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 94;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 96;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 99;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 38;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 29;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 32;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 35;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 41 ;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 62;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 46;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 49;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 103;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 99;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 113;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 114;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 115;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 116;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 117;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 118;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 102;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 119;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 104;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98; (d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 99;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 105;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 99;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 106;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 107;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 108;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 109;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 99;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 110;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 2;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; (d) a VL CDRl having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDRl having the amino acid sequence of SEQ ID NO: 111 ;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 95;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 3;

(d) a VL CDRl having the amino acid sequence of SEQ ID NO: 7;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 8; and

(a) a VH CDRl having the amino acid sequence of SEQ ID NO: 112;

(b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 97;

(c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 98;

(d) a VL CDRl having the amino acid sequence of SEQ ID NO: 99;

(e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 100; and

(f) a VL CDR3 having the amino acid sequence of SEQ ID NO: 9. In one embodiment, these anti-KIT antibodies, or antigen-binding fragments thereof, have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein. a. CDRls

Based on the structural studies performed by the inventors, it has been surprisingly discovered that the VH CDRl and VL CDRl domains of the antibodies of the invention play an important role in the binding specificity/affinity of an antibody, or antigen-binding portion thereof, of the invention for the KIT antigen (see Examples 1-9, below).

Accordingly, in one embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to any one of SEQ ID NOs: 1, 13, 16, 19, 24, 63, 65, 94, 96, 101, 102, 104, 105, 106, 107, 108, 109, 110, 111 or 112. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 1. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 13. The anti- KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 16. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 19. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 24. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 63. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 65. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 94. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 96. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 101. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 102. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 104. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 105. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 106. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 107. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 108. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 109. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 110. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 111. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to SEQ ID NO: 112. In one embodiment, these anti-KIT antibodies, or antigen-binding portions thereof, may have at least two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In another embodiment, the invention comprises anti-KIT antibodies, or antigen- binding portions thereof, which comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to any one of SEQ ID NOs: 7, 29, 32, 35, 38, 41, 62, 46, 49, 64, 66, 99, 103, 113, 114, 115, 116, 117, 118 or 119. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 7. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 29. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 32. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 35. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 38. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 41. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 62. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 46. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 49. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 64. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 66. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 99. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 103. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 113. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 114. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 115. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 116. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 117. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 118. The anti-KIT antibodies, or antigen-binding portions thereof, may comprise or have a VL CDRl having an amino acid sequence identical to or comprising 1, 2 or 3 amino acid residue substitutions relative to SEQ ID NO: 119. In one embodiment, these anti-KIT antibodies, or antigen-binding portions thereof, may have two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

In another embodiment, the present invention provides an anti-KIT antibody, or antigen -binding portion thereof, which comprises or has:

(a) a VH CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to any one of SEQ ID NOs: 1, 13, 16, 19, 24, 63, 65, 94, 96, 101, 102, 104, 105, 106, 107, 108, 109, 110, 111 or 112; and

(b) a VL CDRl having an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue substitutions relative to any one of SEQ ID NOs: 7, 29, 32, 35, 38, 41, 62, 46, 49, 64, 66, 99, 103, 113, 114, 115, 116, 117, 118 or 119.

The invention contemplates antibodies, and antigen-binding antibody fragments thereof, having any combination of the foregoing VH and VL CDRls. For example, antibodies comprising:

(a) a VH CDRl having a sequence identical to SEQ ID NO: 19,

(b) a VL CDRl having an amino acid sequence comprising one amino acid substitution relative to SEQ ID NO: 7. All other combinations are similarly contemplated. In certain embodiments, the antibodies, or antibody fragments thereof, are human antibodies that bind to human KIT and inhibit KIT activity, such as antibodies which block homotypic interactions between Arg381 and Glu386 of the D4 domain of KIT, inhibit SCF- stimulated autophosphorylation of KIT, or inhibit SCF-stimulated cell proliferation.

In one embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise or have:

a VH CDR1 having the amino acid sequence Χ' 1ΥΧ2ΈΗ, wherein X 1¹ is one or zero amino acid residues, and X is selected from the group consisting of S and M (SEQ ID NO:63);

a VH CDR2 comprising the amino acid sequence of SIYPYSGYTYYADSVKG (SEQ ID NO:2); and

a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3). In one embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise or have:

a VH CDR1 having the amino acid sequence

wherein X 1¹ is selected from the group consisting of S and D, and X is selected from the group consisting of S, V and A (SEQ ID NO: 120);

a VH CDR2 comprising the amino acid sequence of YPYSGY (SEQ ID NO:95); and a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3). In one embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise or have:

a VH CDR1 having the amino acid sequence

X 1¹ is selected from the group consisting of S and D; X is selected from the group consisting of S, V and A; and X is selected from the group consisting of S and M (SEQ ID NO: 121);

a VH CDR2 comprising the amino acid sequence of IYPYSGYT (SEQ ID NO:97); and

a VH CDR3 comprising the amino acid sequence of AR YVYHALDY (SEQ ID NO:98).

In another embodiment, the anti-KIT antibodies, or antigen-bindingn portions thereof, of the invention, comprise or have:

a VL CDR1 having the amino acid sequence RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein

X 1 is selected from the group consisting of S, I, R, P and N; X 2 is selected from the group consisting of V, R, Y G and F; X is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R, V and T, X⁵ is zero or one amino acid residues, X⁶ is zero or one amino acid residues, and X⁷ is zero or one amino acid residues (SEQ ID NO:64);

a VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO: 8); and a VL CDR3 having the amino acid sequence QQWAVHSLIT (SEQ ID NO:9).

In yet another embodiment, the anti-KIT antibodies, or antigen-binding portions thereof, of the invention comprise or have:

a VH CDR2 comprising the amino acid sequence of SIYPYSGYTYYADSVKG (SEQ ID NO:2);

a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3); a VL CDR1 having the amino acid sequence RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein

In another embodiment, the invention provides an antibody, or antigen-binding portion thereof, which comprises or has:

a VH CDR1 having the amino acid sequence Χ' 1ΥΧ2ΈΗ, wherein X 1¹ is an amino acid selected from the group consisting of no amino acid, S and V, and X is selected from the group consisting of S and M (SEQ ID NO:65);

a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3). In another embodiment, the invention provides an antibody, or antigen-binding portion thereof, which comprises or has:

X 1 is selected from the group consisting of S, I, R, P and N; X 2 is selected from the group consisting of V, R, Y G and F; X is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R, V and T, X⁵ is selected from the group consisting of no amino acid, L, R, N, S, V and R, X⁶ is selected from the group consisting of no amino acid, R, P, V and I, and X is selected from the group consisting of no amino acid, R, M or S (SEQ ID NO:66);

In yet another embodiment, the invention provides an antibody, or antigen-binding portion thereof, which comprises or has:

a VH CDR1 having the amino acid

a VH CDR2 comprising the amino acid sequence of YPYSGY (SEQ ID NO:95); and a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3); a VL CDR1 having the amino acid sequence RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein

a VH CDR1 having the amino acid

wherei ·n X 1¹ is selected from the group consisting of S and D; X is selected from the group consisting of S, V and A; and X is selected from the group consisting of S and M (SEQ ID NO: 121),

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence selected from the group consisting of 99,

103, 113, 114, 115, 116, 117, 118 and 119,

a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9). In yet another embodiment, the invention provides an antibody, or antigen-binding portion thereof, which comprises or has:

a VH CDR1 having the amino acid

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QSVSSA (SEQ ID NO:99),

a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

a VH CDR1 having the amino acid

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QRGLRNVA (SEQ ID NO: 103), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

a VH CDR1 having the amino acid

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QIRHRLRRA (SEQ ID NO: 113), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

In yet another embodiment, the invention provides an antibody, or antigen-binding portion thereof, which comprises or has: a VH CDR1 having the amino acid

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QIRKVA (SEQ ID NO: 114), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

a VH CDR1 having the amino acid

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QRYNTRPMA (SEQ ID NO: 115), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

a VH CDR1 having the amino acid

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QRGRTA (SEQ ID NO: 116), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

a VH CDR1 having the amino acid

wherei ·n X 1¹ is selected from the group consisting of S and D; X is selected from the group consisting of S, V and A; and X is selected from the group consisting of S and M (SEQ ID NO: 121), a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QRGARSA (SEQ ID NO: 1 17), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

1 2 3 · 1

a VH CDR1 having the amino acid sequence GFNB X^{^}YX", wherein X¹ is selected from the group consisting of S and D; X is selected from the group consisting of S, V and A; and X is selected from the group consisting of S and M (SEQ ID NO: 121),

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QPFRRVA (SEQ ID NO: 118), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9).

1 2 3 · 1

a VH CDR2 having the amino acid sequence IYPYSGYT (SEQ ID NO: 97), a VH CDR3 having the amino acid sequence ARYVYHALDY (SEQ ID NO: 98), a VL CDR1 having an amino acid sequence QNGNVRISA (SEQ ID NO: 119), a VL CDR2 having the amino acid sequence SAS (SEQ ID NO: 100), and

a VL CDR3 having the amino acid sequence of QQWAVHSLIT (SEQ ID NO:9). See, e.g., Tables 1, 2 and 4 for a representation of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences encompassed by the present invention which can be present in any combination to form an anti-KIT antibody, or antigen-binding portion thereof. Exemplary antibodies of the invention have such sequence (structure), bind specifically to human KIT, and have at least two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the biological characteristics described herein.

The present invention encompasses anti-KIT antibodies comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, He, Asn, Gin, Phe, Cys, Pro, Trp, Tyr, Val).

The foregoing applies equally to anti-KIT antibodies and antibody fragments of the invention. Antibodies and antibody fragments having any one or more of the foregoing functional and structural characteristics are contemplated.

3. Framework regions

The variable domains of the heavy and light chains each comprise four framework regions (FR1, FR2, FR3, FR4), which are the more highly conserved portions of the variable domains. The four FRs of the heavy chain are designated FR-H1, FR-H2, FR-H3 and FR- H4, and the four FRs of the light chain are designated FR-Ll, FR-L2, FR-L3 and FR-L4. The Kabat numbering system is one example of a numbering system used herein, See Table 1 and Table 4, Kabat et ah, Supra. As such, Kabat FR-H1 begins at position 1 and ends at approximately amino acid 30, FR-H2 is approximately from amino acid 36 to 49, FR-H3 is approximately from amino acid 66 to 94 and FR-H4 is approximately amino acid 103 to 113. FR-Ll begins at amino acid 1 and ends at approximately amino acid 23, FR-L2 is

approximately from amino acid 35 to 49, FR-L3 is approximately from amino acid 57 to 88 and FR-L4 is approximately from amino acid 98 to 107. In certain embodiments the framework regions may contain substitutions according to the Kabat numbering system, e.g., insertion at 106A in FR-Ll . In addition to naturally occurring substitutions, one or more alterations (e.g., substitutions) of FR residues may also be introduced in an anti-KIT antibody. In certain embodiments, these result in an improvement or optimization in the binding affinity of the antibody for KIT, for example one or more of human, mouse, or cynomolgous KIT. Examples of framework region residues to modify include those which non-covalently bind antigen directly (Amit et ah, Science, 233:747-753 (1986)); interact with/effect the conformation of a CDR (Chothia et al, J. Mol. Biol, 196:901-917 (1987)); and/or participate in the VL-VH interface (US Patent No. 5,225,539). The instant invention also defines the antibodies using Chothia and IMGT numbering systems (see, e.g., Table 4).

In another embodiment the FR may comprise one or more amino acid changes for the purposes of "germlining". For example, the amino acid sequences of selected antibody heavy and light chains are compared to germline heavy and light chain amino acid sequences and where certain framework residues of the selected VL and/or VH chains differ from the germline configuration (e.g., as a result of somatic mutation of the immunoglobulin genes used to prepare the phage library), it may be desirable to "backmutate" the altered framework residues of the selected antibodies to the germline configuration (i.e., change the framework amino acid sequences of the selected antibodies so that they are the same as the germline framework amino acid sequences). Such "backmutation" (or "germlining") of framework residues can be accomplished by standard molecular biology methods for introducing specific mutations (e.g., site-directed mutagenesis; PCR-mediated mutagenesis, and the like). In one embodiment, the variable light and/or heavy chain framework residues are backmutated. In another embodiment, the variable heavy chain of an antibody of the invention is

backmutated. In another embodiment, the variable heavy chain of an antibody of the invention comprises at least one, at least two, at least three, at least four or more

backmutations.

In certain embodiments, the VH of an anti-KIT antibody of the invention may comprise a FR1, FR2, FR3 and/or FR4 that has an amino acid sequence identity with the corresponding framework regions (i.e., FR1 of antibody X as compared to FR1 of antibody Y) of any one or more of the VH chains of the anti-KIT antibodies described herein and set forth in the Sequence Listing, that is from about 65% to about 100%. In one embodiment, the anti-KIT antibodies comprise, or have, a VH FR amino acid sequence (e.g., FR1, FR2, FR3 and/or FR4) having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the corresponding FR of the VH set forth in any one or more of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58. In certain embodiments, the anti-KIT antibodies may comprise a VH FR (e.g., FRl, FR2, FR3 and/or FR4) having an amino acid sequence identical to, or comprising 1, 2 or 3 amino acid substitutions relative to, the corresponding FR of the VH set forth in any one or more of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58. In particular FRl, FR2, FR3 or FR4 of the VH may each have an amino acid sequence identical to, or comprising 1, 2 or 3 amino acid substitutions relative to, the corresponding FRl, FR2, FR3 or FR4 of the VH set forth in any one or more of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58.

In certain embodiments, the VL of an anti-KIT antibody of the invention may comprise a FRl, FR2, FR3 and/or FR4 that has an amino acid sequence identity with the corresponding framework regions (i.e., FRl of antibody X as compared to FRl of antibody Y) of any one or more of the VH chains of the anti-KIT antibodies described herein and set forth in the Sequence Listing, that is from about 65% to about 100%. In one embodiment, the anti-KIT antibodies comprise a VL FR amino acid sequence (e.g., FRl, FR2, FR3 and/or FR4) having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding FR of the VL chains set forth in any one or more of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60.

In certain embodiments, the anti-KIT antibodies may comprise a VL FR (e.g., FRl, FR2, FR3 and/or FR4) having an amino acid sequence identical to, or comprising 1, 2 or 3 amino acid substitutions relative to, the corresponding FR of the VL chains set forth in any one or more of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60. In particular FRl, FR2, FR3 or FR4 of the VL may each have an amino acid sequence identical to, or comprising 1, 2 or 3 amino acid substitutions relative to, the corresponding FR of the VL chains set forth in any one or more of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60.

In a further embodiment, of any of the foregoing, the antibodies of the invention bind human KIT, have frameworks as described above, and have one or more of the biological characteristics described herein.

It should be understood that antibodies defined based on possessing any one or more of the foregoing properties possesses, at least, such one or more properties but may also possess other functional or structural characteristics.

The present invention contemplates antibodies and antibody fragments having any one or more of the functional and structural properties described herein, and further comprising any combination of VH FRl, FR2, FR3, and FR4 regions described herein. Moreover, the invention contemplates antibodies and antibody fragments having any one or more of the functional and structural properties described herein, and further comprising any combination of VL FR1, FR2, FR3, and FR4 regions described herein. The invention also contemplates antibodies and antibody fragments comprising any combination of VL FR1, FR2, FR3, and FR4 regions and VH FR1, FR2, FR3, FR4 regions. In certain embodiments, the antibody is a human, humanized or chimeric antibody.

4. Anti-KIT Antibodies Binding to Specific Epitopes

The present invention is based, at least in part, on structural studies and the crystallization of anti-KIT antibodies, and antigen-binding portions thereof, bound to the D4- D5 domain of human KIT. These studies allowed the inventors of the instant application to identify epitopes, such as conformational epitopes, of the D4 domain of human KIT to which the antibodies, or antigen-binding portions thereof, of the invention may bind.

The antibodies, or antigen-binding portions thereof, of the invention may bind protein motifs or consensus sequences which represent a three dimensional structure in the protein. Such motifs or consensus sequences do not represent a contiguous string of amino acids, but a non-contiguous amino acid arrangement that results from the three-dimensional folding of the KIT receptor (i.e., a "structural motif or "non-linear epitope"). An example of such a motif would be the D4-D4 binding interface of a KIT receptor. In one embodiment, an antibody of the present invention binds to, for example, a non-linear epitope in the D4-D4 interface, preventing the activation of the KIT receptor.

The anti-KIT antibody, or antigen-binding portion thereof, of the invention may bind to the same epitope as Fabl9, Fabl2I, or Fab79D. Alternatively, the anti-KIT antibody, or antigen-binding portion thereof, may bind to the same epitope as an antibody comprising the six CDRs of Fabl9, Fabl2I or Fab79D.

The anti-KIT antibody, or antigen-binding portion thereof, of the invention may bind to amino acid residues Pro317-Asn320 of human KIT. In a preferred embodiment, the antibody, or antigen-binding portion thereof, further binds to amino acid residues Glu 329- Asp332, Ee334, Glu336, Lys358, Glu360, Tyr362, Lys364, Glu366, Arg372, Glu376, His378, Thr380 and Arg381 of human KIT. In another embodiment, the antibody, or antigen-binding portion thereof, further binds to amino acid residues Phe316, Val325, Glu329-Asp332, Ile334, Glu336, Glu360, Tyr362-Lys364, Glu366, Arg372, Glu376, His378, Thr380 and Arg381 of human KIT. The anti-KIT antibody, or antigen-binding portion thereof, of the invention may comprise a heavy chain complementary determining region (CDR) 3 which binds to amino acid residues Glu329, Val331, Asp332, Lys358, Glu360, Glu376, His378 and Thr380 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, may further comprise a heavy chain CDR2 that binds to amino acid residues Pro317, Met318, Asn320, Ile334, Glu336, Lys364 and Arg372 of human KIT. The antibody, or antigen- binding portion thereof, may further comprise a heavy chain CDR1 that binds to amino acid residues Ile319, Asn320 and Glu329-Val331 of human KIT. The antibody, or antigen- binding portion thereof, may further comprise a light chain CDR3 that binds to amino acid residues Tyr362, Glu366 and Arg381 of human KIT. The antibody, or antigen-binding portion thereof, may further comprise a light chain CDR2 that binds to amino acid residues Tyr362, Glu366 and Arg381 of human KIT. The antibody, or antigen-binding portion thereof, may further comprise a light chain CDR1 that does not bind human KIT.

The anti-KIT antibody, or antigen-binding portion thereof, of the invention may comprise a heavy chain complementary determining region (CDR) 3 which binds to amino acid residues Glu329-Asp332, Glu360, Glu376, His378 and Thr380 of human KIT. In one embodiment, the antibody, or antigen-binding portion thereof, may further comprise a heavy chain CDR2 that binds amino acids Phe316-Asn320, Ee334, Glu336, Lys364 and Arg372 of human KIT. In another embodiment, the antibody, or antigen-binding portion thereof, may further comprise a heavy chain CDR1 that binds amino acids Ile319, Asn320, Val325, Asn330 and Glu360 of human KIT. In another embodiment, the antibody, or antigen-binding portion thereof, may further comprise a light chain CDR3 that binds amino acids Tyr362 and Glu376 of human KIT. In another embodiment, the antibody, or antigen-binding portion thereof, may further comprise a light chain CDR2 that binds amino acids Asn330, Thr380 and Arg381 of human KIT. In another embodiment, the antibody, or antigen-binding portion thereof, may further comprise a light chain CDR1 that binds amino acids Glu360, Pro363, Lys364 and Glu366 of human KIT.

It is to be understood that, in certain embodiments, when reference is made to an antibody of the invention binding to an epitope, e.g., a conformational epitope, the intention is for the antibody to bind only to those specific residues that make up the epitope (e.g. , the specific peptides identified) and not other residues in the linear amino acid sequence of the receptor. 5. Nucleotide Sequences Encoding Anti-KIT Antibodies

In addition to the amino acid sequences described above, the present invention further provides nucleotide sequences corresponding to the amino acid sequences and encoding the antibodies of the invention. In one embodiment, the present invention provides

polynucleotides comprising a nucleotide sequence encoding an anti-KIT antibody described herein or fragments thereof. These include, but are not limited to, nucleotide sequences that code for the above referenced amino acid sequences. Thus, the present invention provides polynucleotide sequences encoding VH and VL domain regions including CDRs and FRs of antibodies described herein as well as expression vectors for their efficient expression in cells (e.g., mammalian cells). Methods of making the anti-KIT antibodies using polynucleotides are described below in more detail and are known in the art. The foregoing polynucleotides encode anti-KIT antibodies having the structural and/or functional features described herein. For example, such antibodies bind to human KIT, or a fragment thereof, such as the D4 domain of KIT.

The present invention also encompasses polynucleotides that hybridize under stringent hybridization conditions, e.g., as defined herein, to polynucleotides that encode an antibody of the invention.

Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2X SSC/0.1% SDS at about 50-65°C, highly stringent conditions such as hybridization to filter-bound DNA in 6X SSC at about 45°C followed by one or more washes in 0.1X SSC/0.2% SDS at about 65°C, or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F.M. et ah, eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

In certain embodiments, the polynucleotide sequences of the invention may also comprise a nucleotide sequence encoding an anti-KIT antibody VH that hybridizes under stringent hybridization conditions to the nucleotide sequence of any one of SEQ ID NOs: 67 or 73-79 and/or any of the particular anti-KIT antibody VH sequences provided in the tables and Sequence Listing. In another embodiment, the polynucleotide sequences of the invention may also comprise a nucleotide sequence encoding an anti-KIT antibody VL that hybridizes under stringent conditions to the nucleotide sequence of any one of SEQ ID NOs: 69 or 80-87 and/or any of the particular anti-KIT antibody VL sequences provided in the tables and Sequence Listing.

In certain embodiments, the polynucleotide sequences of the invention may also comprise a nucleotide sequence encoding an anti-KIT antibody VH which has at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of any one of SEQ ID NOs: 67 or 73-79 and/or any of the particular anti-KIT antibody VH sequences provided in the tables and Sequence Listing.

In certain embodiments, the polynucleotide sequences of the invention may also comprise a nucleotide sequence encoding an anti-KIT antibody VL which has at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of any one of SEQ ID NOs: 69 or 80-87 and/or any of the particular anti-KIT antibody VL sequences provided in the tables and Sequence Listing.

The polynucleotides may be obtained, and the nucleotide sequence of the

polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et ah, BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

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

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et ah, 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et ah, eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

6. Biological Characteristics of the Anti-KIT Antibodies

The anti-KIT antibodies of the invention, or antigen-binding fragments thereof, are characterized by, or may have one or more of the biological characteristics described herein. As used herein, the term "biological characteristics" of an antibody refers to any one of the biochemical, binding and functional characteristics, which are used to select antibodies for therapeutic, research, and diagnostic uses as described below.

The biochemical characteristics of the antibodies of the invention include, but are not limited to, isoelectric point (pi) and melting temperature (Tm). The binding characteristics of the antibodies of the invention include, but are not limited to, binding specificity, dissociation constant (Kd), or its inverse, association constant (Ka), or its component k_on or k_Qff rates, epitope, ability to distinguish between various forms and/or preparations of KIT {e.g., recombinant, native, acetylated) and ability to bind soluble and/or immobilized antigen. The functional characteristics of the antibodies of the present invention include, but are not limited to; inhibition of KIT activity, ability to bind active KIT preferentially, ability to bind to the D4 domain of human KIT, cross-reactivity with KIT from one or more non-human species, lack of cross-reactivity with KIT from other species, etc. Methods for measuring the characteristics of the antibodies are well known in the art, some of which are detailed below and in the examples. a. Biochemical Characteristics

Antibodies like all polypeptides have an Isoelectric Point (pi), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein. As used herein the pi value is defined as the pi of the predominant charge form. The pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et ah, 1993, Electrophoresis 14: 1023). In addition, the thermal melting temperatures (Tm) of the Fab domain of an antibody, can be a good indicator of the thermal stability of an antibody and may further provide an indication of the shelf-life. A lower Tm indicates more aggregation/less stability, whereas a higher Tm indicates less aggregation/ more stability. Thus, in certain embodiments antibodies having higher Tm are preferable. Tm of a protein domain {e.g., a Fab domain) can be measured using any standard method known in the art, for example, by differential scanning calorimetry (see, e.g., Vermeer et ah, 2000, Biophys. J. 78:394-404; Vermeer et al, 2000, Biophys. J. 79: 2150-2154).

Accordingly, in certain embodiments the present invention includes anti-KIT antibodies that have certain preferred biochemical characteristics such as a particular isoelectric point (pi) or melting temperature (Tm).

More specifically, in one embodiment, the anti-KIT antibodies of the present invention have a pi ranging from 5.5 to 9.5, e.g., about 5.5 to about 6.0, or about 6.0 to about 6.5, or about 6.5 to about 7.0, or about 7.0 to about 7.5, or about 7.5 to about 8.0, or about 8.0 to about 8.5, or about 8.5 to about 9.0, or about 9.0 to about 9.5. In other specific

embodiments, the anti-KIT antibodies of the present invention have a pi that ranges from 5.5- 6.0, or 6.0 to 6.5, or 6.5 to 7.0, or 7.0-7.5, or 7.5-8.0, or 8.0-8.5, or 8.5-9.0, or 9.0-9.5. Even more specifically, the anti-KIT antibodies of the present invention have a pi of at least 5.5, or at least 6.0, or at least 6.3, or at least 6.5, or at least 6.7, or at least 6.9, or at least 7.1, or at least 7.3, or at least 7.5, or at least 7.7, or at least 7.9, or at least 8.1, or at least 8.3, or at least 8.5, or at least 8.7, or at least 8.9, or at least 9.1, or at least 9.3, or at least 9.5. In other specific embodiments, the anti-KIT antibodies of the present invention have a pi of at least about 5.5, or at least about 6.0, or at least about 6.3, or at least about 6.5, or at least about 6.7, or at least about 6.9, or at least about 7.1, or at least about 7.3, or at least about 7.5, or at least about 7.7, or at least about 7.9, or at least about 8.1, or at least about 8.3, or at least about 8.5, or at least about 8.7, or at least about 8.9, or at least about 9.1, or at least about 9.3, or at least about 9.5.

It is possible to optimize solubility by altering the number and location of ionizable residues in the antibody to adjust the pi. For example the pi of a polypeptide can be manipulated by making the appropriate amino acid substitutions {e.g., by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine). Without wishing to be bound by any particular theory, amino acid substitutions of an antibody that result in changes of the pi of the antibody may improve solubility and/or the stability of the antibody. One skilled in the art would understand which amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pi. In one embodiment, a substitution is generated in an antibody of the invention to alter the pi. It is specifically contemplated that the substitution(s) of the Fc region that result in altered binding to FcyR (described supra) may also result in a change in the pi. In another embodiment,

substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in FcyR binding and any desired change in pi.

In one embodiment, the ant-KIT antibodies of the present invention have a Tm ranging from 65°C to 120°C. In specific embodiments, the ant-KIT antibodies of the present invention have a Tm ranging from about 75°C to about 120°C, or about 75°C to about 85°C, or about 85°C to about 95°C, or about 95°C to about 105°C, or about 105°C to about 115°C, or about 115°C to about 120°C. In other specific embodiments, the ant-KIT antibodies of the present invention have a Tm ranging from 75°C to 120°C, or 75°C to 85°C, or 85°C to 95°C, or 95°C to 105°C, or 105°C to 115°C, or 115°C to 120°C. In still other specific

embodiments, the ant-KIT antibodies of the present invention have a Tm of at least about 65°C, or at least about 70°C, or at least about 75°C, or at least about 80°C, or at least about 85°C, or at least about 90°C, or at least about 95°C, or at least about 100°C, or at least about 105°C, or at least about 1 10°C, or at least about 115°C, or at least about 120°C. In yet other specific embodiments, the ant-KIT antibodies of the present invention have a Tm of at least 65°C, or at least 70°C, or at least 75°C, or at least 80°C, or at least 85°C, or at least 90°C, or at least 95°C, or at least 100°C, or at least 105°C, or at least 110°C, or at least 115°C, or at least 120°C. b. Binding Characteristics

As described above, the anti-KIT antibodies of the invention bind at least one epitope or antigenic determinant of the human KIT receptor or fragment thereof (e.g., the D4 domain of the human KIT receptor) either exclusively or preferentially with respect to other polypeptides. The term "epitope" is defined above, and includes a protein determinant capable of binding to an antibody. These protein determinants or epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The term "discontinuous epitope" as used herein, refers to a conformational epitope on a protein antigen which is formed from at least two separate regions in the primary sequence of the protein.

In certain embodiments, the anti-KIT antibodies bind to human KIT and antigenic fragments thereof. In one embodiment, the anti-KIT antibodies specifically bind to the membrane proximal region of KIT. In certain embodiments, the anti-KIT antibodies bind the D4 domain of KIT. In certain embodiments, the anti-KIT antibodies bind the same epitope as an antibody comprising the six CDRs of any of the antibodies listed in the Examples or Sequence Listing.

The antibodies of the invention may bind epitopes conserved across species. For example, antibodies of the invention may bind murine, non-human primate, rat, bovine, pig or other mammalian KIT and antigenic fragments thereof. In one embodiment, the antibodies of the invention may bind to one or more KIT orthologs and or isoforms. In a specific embodiment, antibodies of the invention may bind to ΚΓΓ and antigenic fragments thereof from one or more species, including, but not limited to, mouse, rat, monkey, primate, and human. In certain embodiments, the antibodies of the invention may bind an epitope within humans across KIT homologs and/or isoforms and/or conformational variants and/or subtypes.

The interactions between antigens and antibodies are the same as for other non- covalent protein-protein interactions. In general, four types of binding interactions exist between antigens and antibodies: (i) hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forces between Lewis acids and Lewis bases, and (iv) hydrophobic interactions. Hydrophobic interactions are a major driving force for the antibody-antigen interaction, and are based on repulsion of water by non-polar groups rather than attraction of molecules (Tanford, 1978). However, certain physical forces also contribute to antigen-antibody binding, for example, the fit or complimentary of epitope shapes with different antibody binding sites. Moreover, other materials and antigens may cross-react with an antibody, thereby competing for available free antibody.

Measurement of the affinity constant and specificity of binding between antigen and antibody is a pivotal element in determining the efficacy of therapeutic, diagnostic and research methods using the anti-KIT antibodies. "Binding affinity" generally refers to the strength of the sum total of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (Kd), which is calculated as the ratio k_0ff/k_on. See, e.g., Chen, Y., et ah, (1999) J. Mol Biol 293:865-881. Affinity can be measured by common methods known in the art, including those described and exemplified herein, such as BIACORE™. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.

The present anti-KIT antibodies have binding affinities for a KIT epitope that include a dissociation constant (K_d) of less than lxlO^"2M, lxlO^"3M, lxlO^"4M, lxlO^"5M, lxlO^"6M, lxlO^"7M, lxlO^"8M, lxlO^"9M, lxlO^"10M, lxlO^"uM, lxlO^"12M, lxlO^"13M, lxlO^"14M or less

15 7 than lxlO^"1JM. In one embodiment, the anti-KIT antibodies have a K_d of less than 10^" M, less than 5xlO^"8M, less than 10^"8M, less than 5xlO^"9M, less than 10^"9M, less than 5xlO^"10M, less than 10^"10M, less than 5xlO^"uM, less than 10^"UM, less than 5xlO^"12M, less than 10^"12M, less than 5xlO^"13M, less than 10^"13M, less than 5xlO^"14M, less than 10^"14M, less than 5xlO^"15M, or less than 10^"15 M. In certain embodiments, anti-KIT antibodies have binding affinities for a KIT epitope that include a dissociation constant (K_d) of between lxlO^~6M and lxlO^~10M, lxlO^"6M and lxlO^"uM, lxlO^"6M and lxlO^"12M, lxlO^"6M and lxlO^"13M, lxlO^"6M and lxlO^"15M, lxlO^"6M and lxlO^"15M, lxlO^"7M and lxlO^"10M, lxlO^"7M and lxlO^"uM, lxlO^"7M and lxlO^"12M, lxlO^"7M and lxlO^"13M, lxlO^"7M and lxlO^"14M, lxlO^"7M and lxlO^"15M, lxlO^"8M and lxlO^"10M, lxlO^"8M and lxlO^"uM, lxlO^"8M and lxlO^"12M, lxlO^"8M and lxlO^"13M, lxlO^"8M and lxlO^"14M, lxlO^"8M and lxlO^"15M, lxlO^"9M and lxlO^"10M, lxlO^"9M and lxlO^"uM, lxlO^"9M and lxlO^"12M, lxlO^"9M and lxlO^"13M, lxlO^"9M and lxlO^"14M and lxlO^"9M and lxlO^"15M. In certain embodiments, K_d is measured by BIACORE™. In certain embodiments, K_d is measured by cell binding.

In certain embodiments, the anti- KIT antibodies are high-affinity antibodies. As used herein, the term "high affinity", when referring to an IgG type antibody, refers to an antibody having a KD of 10^"8 M or less, more preferably 10^"9 M or less and even more preferably 10^"10 M or less for an antigen, e.g., the D4 domain of KIT. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype

-7 -8

refers to an antibody having a K_D of 10^" M or less, more preferably 10^" M or less, even more preferably 10^"9 M or less for an antigen, e.g., the D4 domain of ΚΓΓ. In certain embodiments, the anti-KIT antibodies have an affinity between 5 pM and 200 pM for active human KIT, as assessed by plasmon resonance. In certain embodiments, the affinity is approximately 5, 10, 15, 20, 25, 50, 60, 70 ,75, 80, 90, 100, etc. pM. In certain embodiments, the affinity is between about 5 pM and 50 pM. In certain embodiments, the affinity is between about 5 pM and 100 pM.

In certain embodiments, the anti-KIT antibodies are described as having a binding affinity of a specific molarity or better. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, for an antibody which has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.

In an alternative embodiment, the affinity of the anti-KIT antibodies is described in terms of the association constant (K_a), which is calculated as the ratio k_on/k₀ff. In this instance the present anti-KIT antibodies have binding affinities for a KIT epitope that include an association constant (K_a) of at least lxl0²M⁴, lxl0³M⁴, lxl0⁴M⁴, lxl0⁵M⁴, lxl0⁶M⁴, lxl0⁷M⁴, lxl0⁸M⁴, lxl0⁹M⁴, lxl0^loM⁴ lxl0^uM⁴ lxl0¹²M⁴, lxl0¹³M⁴, lxl0¹⁴M⁴ or at least lxl0¹⁵M⁴. In one embodiment, the anti-KIT antibodies have a K_a of at least 10⁷ M⁴, at least 5 X 10⁷ M⁴, at least 10⁸ M^"1, at least 5 X 10⁸ M^"1, at least 10⁹ M⁴, at least 5 X 10⁹ M⁴, at least 10¹⁰ M⁴, at least 5 X 10¹⁰ M⁴, at least 10¹¹ M⁴, at least 5 X 10¹¹ M⁴, at least 10¹² M⁴, at least 5 X 10¹² M⁴, at least 10¹³ M⁴, at least 5 X 10¹³ M⁴, at least 10¹⁴ M⁴, at least 5 X 10¹⁴ M⁴, at least 10¹⁵ M⁴, or at least 5 X 10¹⁵ M⁴. In certain embodiments, anti-KIT antibodies have binding affinities for a KIT epitope that include an association constant (K_a) of between lxl0²M⁴ and 1χ10³Μ⁴, lxl0²M⁴ and 1χ10⁴Μ⁴, lxl0²M⁴ and 1χ10⁵Μ⁴, lxl0²M⁴ and lxl0⁶M⁴, lxl0³M⁴ and 1χ10⁴Μ⁴, lxl0³M⁴ and 1χ10⁵Μ⁴, lxl0³M⁴ and lxl0⁶M⁴, lxl0⁴M⁴ and 1χ10⁵Μ⁴, lxl0⁴M⁴ and lxl0⁶M⁴ and lxl0⁵M⁴ and lxl0⁶M⁴.

In certain embodiments the rate at which the anti-KIT antibodies dissociate from a KIT epitope may be more relevant than the value of the K_d or the K_a. In this instance the anti- KIT antibodies of the invention may bind to KIT, or a fragment thereof, with a k_Qff of less than 10^"2 s⁴, less than 10^"3 s⁴, less than 5x10^~3 s⁴, less than 10^"4 s⁴, less than 5x10^~4 s⁴, less than 10^"5 s⁴, less than 5x10^~5 s⁴, less than 10^"6 s⁴, less than 5x10^~6 s⁴, less than 10^"7 s⁴, less than 5x10^~7 s⁴, less than 10^"8 s⁴, less than 5x10^~8 s⁴, less than 10^"9 s⁴, less than 5x10^~9 s⁴, or less than 10⁴⁰ s⁴. In certain other embodiments the rate at which the anti- KIT antibodies associate with a KIT epitope may be more relevant than the value of the K_d or the K_a. In this instance the anti-KIT antibodies of the invention may bind to KIT, or a fragment thereof, with a kon rate of at least 10⁵ M^'V¹, at least 5xl0⁵ M^_1s^_1, at least 10⁶ M^_1s^_1, at least 5 x 10⁶ M^" ¹ s^"1 , at least 10⁷ M^"1 s^"1 , at least 5 x 10⁷ M^"1 s^"1 , or at least 10⁸ M^"1 s^"1 , or at least 10⁹ M^"1 s^"1.

Determination of binding affinity can be measured using the specific techniques described further in the Example section, and methods well known in the art. One such method includes measuring the disassociation constant "Kd" by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay that measures solution binding affinity of Fabs for antigen by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti- Fab antibody-coated plate (Chen, et al, (1999) J. Mol Biol 293:865-881). To establish conditions for the assay, microtiter plates (Dynex) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (H 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C). In a non-adsorbant plate (Nunc #269620), 100 pM or 26 pM r 125^JI]-anti gen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res.

57:4593-4599). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., 65 hours) to insure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% Tween- 20 in PBS. When the plates have dried, 150 μΐ/well of scintillant (MicroScint-20; Packard) is added, and the plates are counted on a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

The Kd value may also be measured by using surface plasmon resonance assays using a BIACORE™-2000 or a BIACORE™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ^~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 110 mM sodium acetate, pH 4.8, into 5 ug/ml (^~0.2 uM) before injection at a flow rate of 5 ul/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, IM ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25° C at a flow rate of approximately 25 ul/min. Association rates (k_on) and dissociation rates (k_Qff) are calculated using a simple one-to-one Langmuir binding model (BIACORE™ Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgram.

If the on-rate exceeds 10⁶ M^-1 S^-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette. An "on-rate" or "rate of association" or "association rate" or "k_on" according to this invention can also be determined with the same surface plasmon resonance technique described above using a BIACORE™- 2000 or a BIACORE™-3000 (BIAcore, Inc., Piscataway, N.J.) as described above.

Methods and reagents suitable for determination of binding characteristics of an antibody of the present invention, or an altered/mutant derivative thereof (discussed below), are known in the art and/or are commercially available (see, for example, U.S. Patent Nos. 6,849,425; 6,632,926; 6,294,391 ; 6, 143,574). Moreover, equipment and software designed for such kinetic analyses are commercially available (e.g., BIACORE^® A100, and BIACORE ^® 2000 instruments; Biacore International AB, Uppsala, Sweden).

In one embodiment, a binding assay may be performed either as a direct binding assay or as a competition-binding assay. Binding can be detected using standard ELISA or standard Flow Cytometry assays. In a direct binding assay, a candidate antibody is tested for binding to KIT antigen. Competition-binding assays, on the other hand, assess the ability of a candidate antibody to compete with a known anti-KIT antibody or other compound that binds KIT. In general, any method that permits the detection and/or measuring of binding of an antibody with KIT antigen may be used for detecting and measuring the binding

characteristics of the antibodies of the invention. One of skill in the art will recognize these well known methods and for this reason are not provided in detail here. c. Functional Characteristics

In certain embodiments, the anti-KIT antibodies of the invention modulate, e.g., inhibit, human ΚΓΓ activity, such as blocking the homotypic interactions between Arg381 and Glu386 of human KIT, inhibiting SCF-stimulated autophosphorylation of KIT, and/or inhibiting SCF-stimulated cell proliferation. These functional characteristics can be measured in cell-based or cell free systems, as set forth in the Examples. In one embodiment, the anti-KIT antibodies of the invention neutralize a biological activity of KIT, particularly human KIT.

Neutralization assays are performed using methods known in the art, some of which are described in the examples. The ability of the antibodies of the invention to neutralize of KIT, such as human KIT, may be reflected with an IC50 of lxlO^"6 M or less, lxlO^"7 M or less, lxlO^"8 M or less, lx lO^"9 M or less, lxlO^"10 M or less and lxlO^"11 M or less. In certain embodiments, the neutralization of KIT is with an IC50 of between lxlO^"6 M and lxlO^"9 M, lxlO^"6 M and lxlO^"10 M, lxlO^"6 M and lxlO^"11 M, lxlO^"7 M and lxlO^"9 M, lxlO^"7 M and lxlO^"10 M, lxlO^"7 M and lxlO^"11 M, lxlO^"8 M and lxlO^"9 M, lxlO^"8 M and lxlO^"10 M and

-8 -11

1x10^" M and 1x10^" M. In a further embodiment, the anti-KIT antibodies neutralize at least one of (one or more of) chimpanzee KIT, baboon KIT, marmoset KIT, cynomolgus KIT, rhesus KIT, rat KIT, mouse KIT, pig KIT or other mammalian KIT. The term "inhibitory concentration 50%" (abbreviated as "IC50") represents the concentration of an inhibitor (e.g., an anti-KIT antibody of the invention) that is required for 50% inhibition of a given activity of the molecule the inhibitor targets (e.g., KIT). It will be understood by one of ordinary skill in the art that a lower IC50 value corresponds to a more potent inhibitor. When anti-KIT antibodies inhibit the activity of human KIT and KIT from one or more additional species, inhibition can be with about the same IC50 value or substantially equipotent IC50 value.

In another embodiment, the anti-KIT antibodies of the invention inhibit one or more biological activities of KIT, such as blocking the homotypic interactions between Arg381 and Glu386 of human KIT, inhibiting SCF-stimulated autophosphorylation of KIT, and/or inhibiting SCF-stimulated cell proliferation. The term "inhibition" as used herein, refers to any statistically significant decrease in biological activity, including full blocking of the activity. For example, "inhibition" can refer to a decrease of about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity. In certain embodiments, the anti-KIT antibodies inhibit one or more biological activities of KIT by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In one embodiment, the anti-KIT antibodies of the invention exhibit a reduced antibody related toxicity as compared to previously described anti-KIT antibodies.

In certain embodiments, the antibodies of the invention may bind epitopes conserved across species. In one embodiment, antibodies of the invention bind murine, non-human primate, rat, bovine, pig or other mammalian KIT and antigenic fragments thereof. In one embodiment the antibodies of the invention may bind to one or more KIT orthologs and or isoforms. In a specific embodiment, antibodies of the invention bind to KIT and antigenic fragments thereof from one or more species, including, but not limited to, mouse, rat, monkey, primate, and human. In certain embodiments, the antibodies of the invention may bind an epitope within humans across KIT homologs and/or isoforms and/or conformational variants and/or subtypes.

Exemplary anti-KIT antibodies are provided herein (See Examples and Tables 1-4, and Sequence Listing). Functional characteristics of these exemplary antibodies are provided in the examples. It is contemplated that antibodies of the invention can be described using any one or more of the sequence (structural) and functional characteristics described herein, including features described in the examples and tables.

In certain embodiments, an antibody of the invention binds to human ΚΓΓ and has one or more of the characteristics set forth in the examples vis-a-vis affinity, specificity, neutralization capacity (inhibition of ΚΓΓ bioactivity), epitope binding, etc. In certain embodiments, an antibody of the invention binds to human KIT and has one or more of the biological characteristics described herein.

II. Production of Anti-KIT Antibodies

The following sectiondescribes exemplary techniques for the production of the antibodies useful in the present invention. Such techniques are merely exemplary. It should be understood that these techniques can be used in the production of antibodies and antibody fragments, where applicable. Throughout this section and the application, reference to antibodies is intended to refer to both antibodies and, where applicable, antibody fragments. Moreover, the invention contemplates anti-KIT antibodies and antibody fragments having any of the features (e.g., tagged, human, etc.) described in this section, as well as the use of such antibodies. Some of these techniques are described further in the Example section.

The KIT antigen to be used for production of antibodies may be human KIT or an antigenic fragment thereof. In particular embodiments, the antigenic fragment of human KIT is a fragment that includes the membrane proximal region. Specifically, the antigenic fragment of human KIT is a fragment that includes the D4 domain of the membrane proximal region of human KIT. Human KIT and KIT fragments can be produced recombinantly in an isolated form from, bacterial or eukaryotic cells using standard recombinant DNA

methodology. KIT can be expressed as a tagged (e.g., an epitope tag) or other fusion protein to facilitate isolation as well as identification in various assays. Antibodies or binding proteins that bind to various tags and fusion sequences are available as elaborated below. Other forms of KIT useful for generating antibodies will be apparent to those skilled in the art.

1. Tags

Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol Cell. Biol, 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al, Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al, Protein Engineering, 3(6):547-553 (1990)]. The FLAG-peptide [Hopp et al, BioTechnology, 6: 1204-1210 (1988)] is recognized by an anti-FLAG M2 monoclonal antibody (Eastman Kodak Co., New Haven, Conn.).

Purification of a protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently attached to agarose (Eastman Kodak Co., New Haven, Conn.). Other tag polypeptides include the KT3 epitope peptide [Martin et al, Science, 255: 192-194 (1992)]; an a-tubulin epitope peptide [Skinner et al, J. Biol. Chem., 266: 15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al, Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

2. Monoclonal Antibodies

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma (Kohler et al, Nature, 256:495 (1975); Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), recombinant, and phage display technologies, or a combination thereof. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or multiple antigenic sites in the case of multispecific engineered antibodies. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against the same determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. Following is a description of representative methods for producing monoclonal antibodies which is not intended to be limiting and may be used to produce, for example, monoclonal mammalian, chimeric, humanized, human, domain, diabodies, vaccibodies, linear and multispecific antibodies.

It should be noted that antibodies (monoclonal or polyclonal) or antigen binding portions thereof, may be raised to any epitope of the human Kit protein or, to the concensus sequences discussed herein, or to any conformational, discontinuous, or linear epitopes described herein. Several methods known in the art are useful for specifically selecting an antibody or antigen binding fragment thereof that specifically binds a discontinuous epitope of interest. For example, the techniques disclosed in U.S. Publication No. 2005/0169925, the entire contents of which are incorporated herein by reference, allow for the selection of an antibody which binds to two different peptides within a protein sequence. Such methods may be used in accordance with the present invention to specifically target the conformational and discontinuous epitopes disclosed herein. If the conformational epitope is a protein secondary structure, such structures often form easily in smaller peptides (e.g., <50 amino acids). Thus, immunizing an animal with smaller peptides could capture some conformational epitopes. Alternatively, two small peptides which comprise a conformational epitope may be connected via a flexible linker (e.g. , polyglycol, or a stretch of polar, uncharged amino acids). The linker will allow the peptides to explore various interaction orientations. Immunizing with this construct, followed by appropriate screening could allow for identification of antibodies directed to a conformational epitope. In a preferred embodiment, peptides to specific conformational or linear epitopes may be generated by immunizing an animal with a particular domain and subsequently screening for antibodies which bind the epitope of interest. In one embodiment cryoelectron microscopy (Jiang et al. (2008) Nature 451, 1130- 1134; Joachim (2006) Oxford University Press ISBN:0195182189) may be used to identify the epitopes bound by an antibody or antigen binding fragment of the invention. In another embodiment, the protein may be crystallized with the bound antibody or antigen binding fragment thereof and analyzed by X-ray crystallography to determine the precise epitopes that are bound. In addition, epitopes may be mapped by replacing portions of a protein sequence with the corresponding sequences from mouse or another species. Antibodies directed to epitopes of interest will selectively bind the human sequence regions and, thus, it is possible to sequentially map target epitopes. This technique of chimera based epitope mapping has been used successfully to identify epitopes in various settings (see Henriksson and Pettersson (1997) Journal of Autoimmunity. 10(6):559-568; Netzer et al. (1999) J Biol Chem. 1999 Apr 16;274(16): 11267-74; Hsia et al. (1996) Mol. Microbiol. 19, 53-63, the entire contents of which are incorporated herein by reference).

3. Hybridoma Techniques

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In the hybridoma method, mice or other appropriate host animals, such as hamster, are immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro, as is typically done when using hybridoma technology to produce human monoclonal antibodies. After immunization (in vivo or in vitro), lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent or fusion partner, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). In certain embodiments, the selected myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody- producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. In one aspect, the myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, /. Immunol., 133:3001 (1984); and Brodeur et ah, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, affinity tags, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. Exemplary purification methods are described in more detail below.

4. Recombinant DNA Techniques

Methods for producing and screening for specific antibodies using recombinant DNA technology are routine and well known in the art (e.g., US Patent No. 4,816,567). DNA encoding the monoclonal antibodies may be readily isolated and/or sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et ah, Curr. Opinion in Immunol, 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992). As described below for antibodies generated by phage display and humanization of antibodies, DNA or genetic material for recombinant antibodies can be obtained from source(s) other than hybridomas to generate antibodies of the invention.

Recombinant expression of an antibody or variant thereof generally requires construction of an expression vector containing a polynucleotide that encodes the antibody. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., US. Patent Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.

Once the expression vector is transferred to a host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce an antibody. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single-chain antibody of the invention, operably linked to a heterologous promoter. In certain embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

Mammalian cell lines available as hosts for expression of recombinant antibodies are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products.

Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the antibody or portion thereof expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030 and

HsS78Bst cells. In one embodiment, human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. In one embodiment, the human cell line PER.C6. (Crucell, Netherlands) can be used to

recombinantly produce monoclonal antibodies.

Additional cell lines which may be used as hosts for expression of recombinant antibodies include, but are not limited to, insect cells (e.g., Sf21/Sf9, Trichoplusia ni Bti- Tn5bl-4) or yeast cells (e.g., S. cerevisiae, Pichia, US7326681; etc), plants cells (US20080066200); and chicken cells (WO2008142124).

In certain embodiments, antibodies of the invention are expressed in a cell line with stable expression of the antibody. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express the antibody molecule may be generated. Host cells can be transformed with an appropriately engineered vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene.

Following the introduction of the foreign DNA, cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrated the plasmid into their chromosomes to grow and form foci which in turn can be cloned and expanded into cell lines. Methods for producing stable cell lines with a high yield are well known in the art and reagents are generally available commercially.

In certain embodiments, antibodies of the invention are expressed in a cell line with transient expression of the antibody. Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell. It is in fact maintained as an extrachromosomal element, e.g., as an episome, in the cell. Transcription processes of the nucleic acid of the episome are not affected and a protein encoded by the nucleic acid of the episome is produced.

The cell line, either stable or transiently transfected, is maintained in cell culture medium and conditions well known in the art resulting in the expression and production of monoclonal antibodies. In certain embodiments, the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other embodiments, the cell culture media is modified to support increases in both cell growth and biologic protein expression. As used herein, the terms "cell culture medium," "culture medium," and "medium formulation" refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture medium may be optimized for a specific cell culture use, including, for example, cell culture growth medium which is formulated to promote cellular growth, or cell culture production medium which is formulated to promote recombinant protein production. The terms nutrient, ingredient, and component are used interchangeably herein to refer to the constituents that make up a cell culture medium.

In one embodiment, the cell lines are maintained using a fed batch method. As used herein, "fed batch method," refers to a method by which a fed batch cell culture is supplied with additional nutrients after first being incubated with a basal medium. For example, a fed batch method may comprise adding supplemental media according to a determined feeding schedule within a given time period. Thus, a "fed batch cell culture" refers to a cell culture wherein the cells, typically mammalian, and culture medium are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture.

The cell culture medium used and the nutrients contained therein are known to one of skill in the art. In one embodiment, the cell culture medium comprises a basal medium and at least one hydrolysate, e.g., soy-based, hydrolysate, a yeast-based hydrolysate, or a

combination of the two types of hydrolysates resulting in a modified basal medium. In another embodiment, the additional nutrients may include only a basal medium, such as a concentrated basal medium, or may include only hydrolysates, or concentrated hydrolysates. Suitable basal media include, but are not limited to Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-Minimal Essential Medium (a-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHO protein free medium (Sigma) or EX- CELL™ 325 PF CHO Serum-Free Medium for CHO Cells Protein-Free (SAFC Bioscience), and Iscove's Modified Dulbecco's Medium. Other examples of basal media which may be used in the invention include BME Basal Medium (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-Invitrogen (# 31600); see also Dulbecco and Freeman (1959) Virology 8, 396; Smith et al. (1960) Virology 12, 185. Tissue Culture Standards Committee, In vitro 6:2, 93); CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker R. C. et al (1957) Special Publications, N.Y. Academy of Sciences, 5, 303).

In certain embodiments, the basal medium may be is serum-free, meaning that the medium contains no serum {e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to one skilled in the art) or animal protein free media or chemically defined media. The basal medium may be modified in order to remove certain non-nutritional components found in standard basal medium, such as various inorganic and organic buffers, surfactant(s), and sodium chloride. Removing such components from basal cell medium allows an increased concentration of the remaining nutritional components, and may improve overall cell growth and protein expression. In addition, omitted components may be added back into the cell culture medium containing the modified basal cell medium according to the requirements of the cell culture conditions. In certain embodiments, the cell culture medium contains a modified basal cell medium, and at least one of the following nutrients, an iron source, a recombinant growth factor; a buffer; a surfactant; an osmolarity regulator; an energy source; and non-animal hydrolysates. In addition, the modified basal cell medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. In another embodiment, the modified basal medium further contains glutamine, e.g., L- glutamine, and/or methotrexate.

In certain embodiments, antibody production is conducted in large quantity by a bioreactor process using fed-batch, batch, perfusion or continuous feed bioreactor methods known in the art. Large-scale bioreactors have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These bioreactors may use agitator impellers to distribute oxygen and nutrients. Small scale bioreactors refers generally to cell culturing in no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters. Alternatively, single-use bioreactors (SUB) may be used for either large-scale or small scale culturing.

Temperature, pH, agitation, aeration and inoculum density will vary depending upon the host cells used and the recombinant protein to be expressed. For example, a recombinant protein cell culture may be maintained at a temperature between 30 and 45 degrees Celsius. The pH of the culture medium may be monitored during the culture process such that the pH stays at an optimum level, which may be for certain host cells, within a pH range of 6.0 to 8.0. An impellor driven mixing may be used for such culture methods for agitation. The rotational speed of the impellor may be approximately 50 to 200 cm/sec tip speed, but other airlift or other mixing/aeration systems known in the art may be used, depending on the type of host cell being cultured. Sufficient aeration is provided to maintain a dissolved oxygen concentration of approximately 20% to 80% air saturation in the culture, again, depending upon the selected host cell being cultured. Alternatively, a bioreactor may sparge air or oxygen directly into the culture medium. Other methods of oxygen supply exist, including bubble-free aeration systems employing hollow fiber membrane aerators.

5. Phage Display Techniques

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al. , Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991). In such methods antibodies of the invention can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries {e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SURFZAP™ phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, US Patent Nos. 6,248,516; US 6,545,142; 6,291,158; 6,291,1591; 6,291,160; 6,291,161; 6,680,192; 5,969,108; 6,172,197; 6,806,079; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,593,081; 6,582,915; 7,195,866. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for generation and isolation of monoclonal antibodies.

In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library {e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, humanized antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et ah, BioTechniques 12(6):864-869 (1992);; and Better et al, Science 240: 1041-1043 (1988).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498. Thus, techniques described above and those well known in the art can be used to generate recombinant antibodies wherein the binding domain, e.g., ScFv, was isolated from a phage display library.

6. Antibody Purification and Isolation

Once an antibody molecule has been produced by recombinant or hybridoma expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography {e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences (referred to herein as "tags") described above or otherwise known in the art to facilitate purification.

When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et ah, Bio/Technology, 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted into the periplasmic space of E. coli. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious

contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and/or affinity chromatography either alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody and will be understood by one of skill in the art. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH₃ domain, the Bakerbond ABX resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion- exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction

chromatography using an elution buffer at a pH between about 2.5-4.5, and performed at low salt concentrations (e.g., from about 0-0.25 M salt).

Thus, in certain embodiments, the present invention provides antibodies that are substantially purified/isolated. In one embodiment, these isolated/purified recombinantly expressed antibodies may be administered to a patient to mediate a prophylactic or therapeutic effect. In another embodiment these isolated/purified antibodies may be used to diagnose a KIT mediated disease.

7. Humanized and Chimeric Antibodies

In certain embodiments, the antibodies and antibody fragments of the invention, including monoclonal antibodies, are humanized antibodies, which are generated using methods well known in the art. Humanized antibodies are antibody molecules derived from a non-human species antibody (also referred to herein as a donor antibody) that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (also referred to herein as an acceptor antibody). Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding and/or reduce immunogenicity. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Riechmann et al., Nature 332:323 (1988)). In practice, and in certain embodiments, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. In alternative embodiments, the FR residues are fully human residues.

Humanization can be essentially performed following the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Supra; Verhoeyen et ah, Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Specifically, humanized antibodies may be prepared by methods well known in the art including CDR grafting approaches (see, e.g., US Patent No. 6,548,640), veneering or resurfacing (US Patent Nos. 5,639,641 and 6,797,492; Studnicka et al, Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS 91:969- 973 (1994)), chain shuffling strategies (see e.g., U.S. Patent No. 5,565,332; Rader et al, Proc. Natl. Acad. Sci. USA (1998) 95:8910-8915), molecular modeling strategies (U.S. Patent No. 5,639,641), and the like. These general approaches may be combined with standard mutagenesis and recombinant synthesis techniques to produce anti-KIT antibodies with desired properties.

CDR grafting is performed by replacing one or more CDRs of an acceptor antibody {e.g., a human antibody) with one or more CDRs of a donor antibody {e.g., a non-human antibody). Acceptor antibodies may be selected based on similarity of framework residues between a candidate acceptor antibody and a donor antibody and may be further modified to introduce similar residues. Following CDR grafting, additional changes may be made in the donor and/or acceptor sequences to optimize antibody binding and functionality.

Grafting of abbreviated CDR regions is a related approach. Abbreviated CDR regions include the specificity-determining residues and adjacent amino acids, including those at positions 27d-34, 50-55 and 89-96 in the light chain, and at positions 31-35b, 50-58, and 95- 101 in the heavy chain. See (Padlan et al. (1995) FASEB J. 9: 133-9). Grafting of specificity- determining residues (SDRs) is premised on the understanding that the binding specificity and affinity of an antibody combining site is determined by the most highly variable residues within each of the CDR regions. Analysis of the three-dimensional structures of antibody- antigen complexes, combined with analysis of the available amino acid sequence data was used to model sequence variability based on structural dissimilarity of amino acid residues that occur at each position within the CDR. Minimally immunogenic polypeptide sequences consisting of contact residues, which are referred to as SDRs, are identified and grafted onto human framework regions.

Veneering or resurfacing is based on the concept of reducing potentially

immunogenic amino acid sequences in a rodent or other non-human antibody by resurfacing the solvent accessible exterior of the antibody with human amino acid sequences. Thus, veneered antibodies appear less foreign to human cells. A non-human antibody is veneered by (1) identifying exposed exterior framework region residues in the non-human antibody, which are different from those at the same positions in framework regions of a human antibody, and (2) replacing the identified residues with amino acids that typically occupy these same positions in human antibodies.

By definition, humanized antibodies are chimeric antibodies. Chimeric antibodies are antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while another portion of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (e.g., Morrison et ah, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a nonhuman primate (e.g., Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

8. Human Antibodies

As an alternative to humanization, human antibodies can be generated using methods well known in the art. Thus, in certain embodiments, the antibodies and antibody fragments, of the invention are human antibodies. Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of functional human antibody loci into a rodent, other mammal or animal so that the rodent, other mammal or animal produces fully human antibodies.

For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (½) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.

5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852. In practice, the use of XENOMOUSE® strains of mice that have been engineered to contain up to but less than 1000 kb-sized germline configured fragments of the human heavy chain locus and kappa light chain locus. See Mendez et al. Nature Genetics 15: 146- 156 (1997) and Green and Jakobovits /. Exp. Med. 188:483-495 (1998). The

XENOMOUSE® strains are available from Amgen, Inc. (Fremont, Calif.).

The production of the XENOMOUSE® strains of mice and antibodies produced in those mice is further discussed and delineated in U.S. Patent Nos. 6,673,986; 7,049,426; 6,833,268; 6,162,963, 6,150,584, 6,114,598, 6,075,181, 6,657,103; 6,713,610 and 5,939,598; US Publication Nos. 2004/0010810; 2003/0229905; 2004/0093622; 2005/0054055;

2005/0076395; and 2006/0040363.

Essentially, XENOMOUSE® lines of mice are immunized with an antigen of interest (e.g., KIT), lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines using techniques described above an well known in the art. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produced antibodies specific to the antigen of interest.

In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more D_H genes, one or more J_H genes, a mu constant region, and usually a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; 5,874,299; 6,255,458; 5,591,669; 6,023,010; 5,612,205; 5,721,367; 5,789,215; 5,643,763; and 5,981,175.

Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See Patent No. 6,632,976. Additionally, KM™— mice, which are the result of cross-breeding of Kirin's Tc mice with Medarex's minilocus (Humab) mice have been generated. These mice possess the human IgH transchromosome of the Kirin mice and the kappa chain transgene of the Genpharm mice (Ishida et ah, Cloning Stem Cells, (2002) 4:91- 102).

Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display (Medlmmune (formerly CAT), Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (Medlmmune (formerly CAT)), yeast display, and the like. The phage display technology (See e.g., US Patent No. 5,969,108) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in- frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single- stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et ah, Nature, 352:624-628 (1991) isolated a diverse array of anti- oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et ah, J. Mol. Biol. 222:581- 597 (1991), or Griffith et al, EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.

5,565,332 and 5,573,905. As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Immunoglobulin genes undergo various modifications during maturation of the immune response, including recombination between V, D and J gene segments, isotype switching, and hyperaiutation in the variable regions. Recombination and somatic

hyperaiutation are the foundation for generation of antibody diversity and affinity maturation, but they can also generate sequence liabilities that may make commercial production of such immunoglobulins as therapeutic agents difficult or increase the immunogenicity risk of the antibody. In general, mutations in CDR regions are likely to contribute to improved affinity and function, while mutations in framework regions may increase the risk of

immunogenicity. This risk can be reduced by reverting framework mutations to germline while ensuring that activity of the antibody is not adversely impacted. The diversification processes may also generate some structural liabilities or these structural liabilities may exist within germline sequences contributing to the heavy and light chain variable domains.

Regardless of the source, it may be desirable to remove potential structural liabilities that may result in instability, aggregation, heterogeneity of product, or increased immunogenicity. Examples of undesirable liabilities include unpaired cysteines (which may lead to disulfide bond scrambling, or variable sulfhydryl adduct formation), N-linked glycosylation sites (resulting in heterogeneity of structure and activity), as well as deamidation (e.g., NG, NS), isomerization (DG), oxidation (exposed methionine), and hydrolysis (DP) sites.

Accordingly, in order to reduce the risk of immunogenicity and improve

pharmaceutical properties of the antibodies of the invention, it may be desirable to revert a framework sequence to germline, revert a CDR to germline, and/or remove a structural liability.

9. Antibody Fragments

In certain embodiments, the antibodies of the invention are antibody fragments or antibodies comprising these fragments. The antibody fragment comprises a portion of the full length antibody, which generally is the antigen binding or variable region thereof.

Examples of antibody fragments include Fab, Fab', F(ab')₂, Fd and Fv fragments. Diabodies; linear antibodies (U.S. Pat. No. 5,641,870); single-chain antibody molecules; and

multispecific antibodies are antibodies formed from these antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies using techniques well known in the art. However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. In one embodiment, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can also be directly recovered from E. coli and chemically coupled to form F(ab')₂ fragments (Carter et ah, Bio/Technology, 10: 163-167 (1992)). According to another approach, F(ab')₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). In certain embodiments, the antibody is not a Fab fragment. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv.

In certain embodiments, the present antibodies are domain antibodies, e.g., antibodies containing the small functional binding units of antibodies, corresponding to the variable regions of the heavy (V_H) or light (V_L) chains of human antibodies. Examples of domain antibodies include, but are not limited to, those available from Domantis that are specific to therapeutic targets (see, for example, WO04/058821; WO04/081026; WO04/003019;

WO03/002609; U.S. Patent Nos. 6,291,158; 6,582,915; 6,696,245; and 6,593,081).

In certain embodiments , the present antibodies of the invention are linear antibodies. Linear antibodies comprise a pair of tandem Fd segments (V_H-C_HI-V_H-C_HI) which form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. See, Zapata et al., Protein Eng., 8(10): 1057-1062 (1995).

10. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the KIT protein. Other such antibodies may combine a KIT binding site with a binding site for another protein. Alternatively, an anti-KIT arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16), so as to focus and localize cellular defense mechanisms to the KIT-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express KIT. These antibodies possess a KIT-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')₂ bispecific antibodies). Methods for making bispecific antibodies are known in the art. (See, for example, Millstein et al., Nature, 305:537-539 (1983); Traunecker et al., EMBO J, 10:3655-3659 (1991); Suresh et al, Methods in Enzymology, 121 :210 (1986); Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992); Hollinger et al., Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993); Gruber et al., J. Immunol., 152:5368 (1994); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; 5,731,168; 4,676,980; 5,897,861; 5,660,827; 5,811,267; 5,849,877; 5,948,647; 5,959,084; 6,106,833; 6,143,873 and 4,676,980, WO 94/04690; and WO 92/20373.)

Traditional production of full length bispecific antibodies is based on the co- expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_H2, and C_H3 regions. It is preferred to have the first heavy-chain constant region (C_HI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.

In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure may facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the C_H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains {e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones {e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end- products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (US Patent No. 5,897,861). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')₂ molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody

heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by HoUinger et al., Proc. Natl. Acad. Sci USA, 90:6444- 6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a V_H connected to a V_Lby a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994) and US Patent Nos. 5,591,828; 4,946,778; 5,455,030; and 5,869,620.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared, Tutt et al. J. Immunol. 147: 60 (1991), and multispecific valencies US Patent No. 5,258,498. 11. Other Amino Acid Sequence Modifications

In addition to the above described human, humanized and/or chimeric antibodies, the present invention also encompasses further modifications and, their variants and fragments thereof, of the anti-KIT antibodies of the invention comprising one or more amino acid residues and/or polypeptide substitutions, additions and/or deletions in the variable light (V_L) domain and/or variable heavy (V_R) domain and/or Fc region and post translational modifications. Included in these modifications are antibody conjugates wherein an antibody has been covalently attached to a moiety. Moieties suitable for attachment to the antibodies include but are not limited to, proteins, peptides, drugs, labels, and cytotoxins. These changes to the antibodies may be made to alter or fine tune the characteristics (biochemical, binding and/or functional) of the antibodies as is appropriate for treatment and/or diagnosis of KIT mediated diseases. Methods for forming conjugates, making amino acid and/or polypeptide changes and post-translational modifications are well known in the art, some of which are detailed below. The following description is not intended to be limiting, but instead a non-limiting description of some embodiments, more of which will be obvious to one of skill in the art. It is also understood that some of the following methods were used to develop the human, humanized and/or chimeric antibody sequences described above. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.

Amino acid changes to the antibodies necessarily results in sequences that are less than 100% identical to the above identified antibody sequences. In certain embodiments, in this context, the antibodies many have about 25% to about 95% sequence identity to the amino acid sequence of either the heavy or light chain variable domain of an anti-KIT antibody as described herein. Thus, in one embodiment a modified antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of an anti-KIT antibody as described herein. In another embodiment, an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain CDR1, CDR2, or CDR3 of an anti-KIT antibody as described herein. In another embodiment, an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain FR1, FR2, FR3 or FR4 of an anti-KIT antibody as described herein.

In certain embodiments, altered antibodies are generated by one or more amino acid alterations (e.g., substitutions, deletion and/or additions) introduced in one or more of the variable regions of the antibody. In another embodiment, the amino acid alterations are introduced in the framework regions. One or more alterations of framework region residues may result in an improvement in the binding affinity of the antibody for the antigen. This may be especially true when these changes are made to humanized antibodies wherein the framework region may be from a different species than the CDR regions. Examples of framework region residues to modify include those which non-covalently bind antigen directly (Amit et ah, Science, 233:747-753 (1986)); interact with/effect the conformation of a CDR (Chothia et al, J. Mol. Biol, 196:901-917 (1987)); and/or participate in the V_L-V_H interface (US Patent Nos. 5,225,539 and 6,548,640). In one embodiment, from about one to about five framework residues may be altered. Sometimes, this may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even where none of the hypervariable region residues have been altered. Normally, however, an altered antibody will comprise additional hypervariable region alteration(s). In certain embodiments, the hypervariable region residues may be changed randomly, especially where the starting binding affinity of an anti-KIT antibody for the antigen from the second mammalian species is such that such randomly produced antibodies can be readily screened.

One useful procedure for generating altered antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, Science, 244: 1081-1085 (1989)). In this method, one or more of the hypervariable region residue(s) are replaced by alanine or polyalanine residue(s) to alter the interaction of the amino acids with the KIT. Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing additional or other mutations at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. The Ala-mutants produced this way are screened for their biological activity as described herein.

In certain embodiments the substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display (Hawkins et ah, J. Mol. Biol., 254:889-896 (1992) and Lowman et ah, Biochemistry, 30(45): 10832-10837 (1991)). Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed mutants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.

Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions yielding fusion proteins, or conservative substitutions of amino acid residues within and/or adjacent to the amino acid sequence, but that result in a "silent" change, in that the change produces a functionally equivalent anti-KIT antibody. Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In addition, glycine and proline are residues that can influence chain orientation. Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, a -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.

In another embodiment, any cysteine residue not involved in maintaining the proper conformation of the anti-KIT antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability

(particularly where the antibody is an antibody fragment such as an Fv fragment).

In certain embodiments of the invention, an antibody can be modified to produce fusion proteins; i.e., the antibody, or a fragment thereof, fused to a heterologous protein, polypeptide or peptide. In certain embodiments, the protein fused to the portion of an antibody is an enzyme component of Antibody-Directed Enzyme Prodrug Therapy (ADEPT). Examples of other proteins or polypeptides that can be engineered as a fusion protein with an antibody include, but are not limited to toxins such as ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed anti- viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell, 47:641 (1986), and Goldenberg et al., Cancer Journal for Clinicians, 44:43 (1994).

Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from

Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232.

Additional fusion proteins may be generated through the techniques of gene-shuffling, motif- shuffling, exon- shuffling, and/or codon- shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the characteristics of the antibody or fragments thereof (e.g., an antibody or a fragment thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721;

5,834,252; and 5,837,458, and Patten et al, 1997, Curr. Opinion Biotechnol, 8:724-33 ; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol., 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313. The antibody can further be a binding-domain immunoglobulin fusion protein as described in U.S. Publication 2003/0118592, and PCT Publication WO 02/056910.

12. Variant Fc Regions

It is known that variants of the Fc region (e.g., amino acid substitutions and/or additions and/or deletions) enhance or diminish effector function of the antibody (See e.g., U.S. Patent Nos. 5,624,821 ; 5,885,573; 6,538,124; 7,317,091 ; 5,648,260; 6,538,124; WO 03/074679; WO 04/029207; WO 04/099249; WO 99/58572; US Publication No.

2006/0134105; 2004/0132101 ; 2006/0008883) and may alter the pharmacokinetic properties (e.g., half-life) of the antibody (see, U.S. patents 6,277,375 and 7,083,784). Thus, in certain embodiments, the anti-KIT antibodies of the invention comprise an altered Fc region (also referred to herein as "variant Fc region") in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of Clq binding and complement dependent cytotoxicity (CDC) or of FcyR binding, for IgG, and antibody- dependent cellular cytotoxicity (ADCC), or antibody dependent cell-mediated phagocytosis (ADCP). The present invention encompasses the antibodies described herein with variant Fc regions wherein changes have been made to fine tune the effector function, enhancing or diminishing, providing a desired effector function. Accordingly, in one embodiment of the invention, the anti-KIT antibodies of the invention comprise a variant Fc region (i.e., Fc regions that have been altered as discussed below). Anti-KIT antibodies of the invention comprising a variant Fc region are also referred to here as "Fc variant antibodies." As used herein native refers to the unmodified parental sequence and the antibody comprising a native Fc region is herein referred to as a "native Fc antibody". Fc variant antibodies can be generated by numerous methods well known to one skilled in the art. Non-limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the antigen-binding portion (e.g., variable regions) of an anti-KIT antibody may be subcloned into a vector encoding a variant Fc region. In one embodiment, the variant Fc region exhibits a similar level of inducing effector function as compared to the native Fc region. In another embodiment, the variant Fc region exhibits a higher induction of effector function as compared to the native Fc. In another embodiment, the variant Fc region exhibits lower induction of effector function as compared to the native Fc. Some specific embodiments of variant Fc regions are detailed infra. Methods for measuring effector function are well known in the art.

The effector function of an antibody is modified through changes in the Fc region, including but not limited to, amino acid substitutions, amino acid additions, amino acid deletions and changes in post translational modifications to Fc amino acids (e.g.,

glycosylation). The methods described below may be used to fine tune the effector function of a present antibody, a ratio of the binding properties of the Fc region for the FcR (e.g., affinity and specificity), resulting in a therapeutic antibody with the desired properties for a particular disease indication and taking into consideration the biology of KIT.

It is understood that the Fc region as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C 2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat. Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index, and thus slight differences between the presented sequence and sequences in the prior art may exist.

In one embodiment, the present invention encompasses Fc variant antibodies which have altered binding properties for an Fc ligand (e.g., an Fc receptor, Clq) relative to a native Fc antibody. Examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K_d), dissociation and association rates (k_0ff and k_on respectively), binding affinity and/or avidity. It is known in the art that the equilibrium dissociation constant (¾) is defined as k₀ff/k_on. In certain aspects, an antibody comprising an Fc variant region with a low K_d may be more desirable to an antibody with a high ¾.

However, in some instances the value of the k_on or k_Qff may be more relevant than the value of the K_d. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. For example, a modification that reduces binding to one or more positive regulator (e.g., FcyRIIIA) and/or enhanced binding to an inhibitory Fc receptor (e.g., FcyRIIB) would be suitable for reducing ADCC activity. Accordingly, the ratio of binding affinities (e.g., the ratio of equilibrium dissociation constants (K_d)) for different receptors can indicate if the ADCC activity of an Fc variant antibody of the invention is enhanced or decreased. Additionally, a modification that reduces binding to Clq would be suitable for reducing or eliminating CDC activity. In one embodiment, Fc variant antibodies exhibit altered binding affinity for one or more Fc receptors including, but not limited to FcRn, FcyRI (CD64) including isoforms FcyRIA, FcyRIB, and FcyRIC; FcyRII (CD32 including isoforms FcyRIIA, FcyRIIB, and FcyRIIC); and FcyRIII (CD16, including isoforms FcyRIIIA and FcyRIIIB) as compared to an native Fc antibody.

In one embodiment, an Fc variant antibody has enhanced binding to one or more Fc ligand relative to a native Fc antibody. In another embodiment, the Fc variant antibody exhibits increased or decreased affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or is between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than a native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for an Fc ligand that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody. In certain embodiments, an Fc variant antibody has increased affinity for an Fc ligand. In other embodiments, an Fc variant antibody has decreased affinity for an Fc ligand.

In a specific embodiment, an Fc variant antibody has enhanced binding to the Fc receptor FcyRIIIA. In another specific embodiment, an Fc variant antibody has enhanced binding to the Fc receptor FcyRIIB. In a further specific embodiment, an Fc variant antibody has enhanced binding to both the Fc receptors FcyRIIIA and FcyRIIB. In certain

embodiments, Fc variant antibodies that have enhanced binding to FcyRIIIA do not have a concomitant increase in binding the FcyRIIB receptor as compared to a native Fc antibody. In a specific embodiment, an Fc variant antibody has reduced binding to the Fc receptor FcyRIIIA. In a further specific embodiment, an Fc variant antibody has reduced binding to the Fc receptor FcyRIIB. In still another specific embodiment, an Fc variant antibody exhibiting altered affinity for FcyRIIIA and/or FcyRIIB has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for FcyRIIIA and/or FcyRIIB has altered binding to Clq relative to a native Fc antibody.

In another embodiment, Fc variant antibodies exhibit affinities for FcyRIIIA receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or are between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than an native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for FcyRIIIA that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody.

In yet another embodiment, Fc variant antibodies exhibit affinities for FcyRIIB receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or are between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than an native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for FcyRIIB that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody.

In one embodiment, Fc variant antibodies exhibit increased or decreased affinities to Clq relative to a native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for Clq receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or are between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 100 fold, or between 75 fold and 200 fold, or between 100 and 200 fold, more or less than an native Fc antibody. In another embodiment, Fc variant antibodies exhibit affinities for Clq that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% more or less than an native Fc antibody. In still another specific embodiment, an Fc variant antibody exhibiting altered affinity for Ciq has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for Clq has altered binding to FcyRIIIA and/or FcyRIIB relative to a native Fc antibody. It is well known in the art that antibodies are capable of directing the attack and destruction of targeted antigen through multiple processes collectively known in the art as antibody effector functions. One of these processes, known as "antibody-dependent cell- mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins.

Specific high-affinity IgG antibodies directed to the surface of target cells "arm" the cytotoxic cells and are required for such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement. Another process encompassed by the term effector function is complement dependent cytotoxicity (hereinafter referred to as "CDC") which refers to a biochemical event of antibody-mediated target cell destruction by the complement system. The complement system is a complex system of proteins found in normal blood plasma that combines with antibodies to destroy pathogenic bacteria and other foreign cells. Still another process encompassed by the term effector function is antibody dependent cell-mediated phagocytosis (ADCP) which refers to a cell- mediated reaction wherein nonspecific cytotoxic cells that express one or more effector ligands recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

It is contemplated that Fc variant antibodies are characterized by in vitro functional assays for determining one or more FcyR mediated effector cell functions. In certain embodiments, Fc variant antibodies have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude Fc variant antibodies that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.

The serum half-life of proteins comprising Fc regions may be increased by increasing the binding affinity of the Fc region for FcRn. The term "antibody half-life" as used herein means a pharmacokinetic property of an antibody that is a measure of the mean survival time of antibody molecules following their administration. Antibody half-life can be expressed as the time required to eliminate 50 percent of a known quantity of immunoglobulin from the patient's body (or other mammal) or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues. Half-life may vary from one immunoglobulin or class of immunoglobulin to another. In general, an increase in antibody half-life results in an increase in mean residence time (MRT) in circulation for the antibody administered.

The increase in half-life allows for the reduction in amount of drug given to a patient as well as reducing the frequency of administration. To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody

(especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. Alternatively, antibodies of the invention with increased half-lives may be generated by modifying amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor (see, for examples, US Patent Nos. 6,821,505 and 7,083,784; and WO 09/058492). In addition, the half-life of antibodies of the invention may be increase by conjugation to PEG or Albumin by techniques widely utilized in the art. In some embodiments antibodies comprising Fc variant regions of the invention have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150% or more as compared to an antibody comprising a native Fc region. In some embodiments antibodies comprising Fc variant regions have an increased half-life of about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or more, or is between 2 fold and 10 fold, or between 5 fold and 25 fold, or between 15 fold and 50 fold, as compared to an antibody comprising a native Fc region.

In certain embodiments the effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein (Claudia Ferrara et al., 2006, Biotechnology and Bioengineering 93:851-861). Thus, glycosylation of the Fc region can be modified to increase or decrease effector function (see for examples, Umana et al, 1999, Nat. Biotechnol 17: 176-180; Davies et al, 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al, 2003, J Biol Chem 278:3466-3473; U.S. Pat. Nos. 6,602,684; 6,946,292; 7,064,191; 7,214,775;7,393,683;

7,425,446; 7,504,256; U.S. Publication. Nos. 2003/0157108; 2003/0003097; 2009/0010921; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland)). Accordingly, in one embodiment the Fc regions of anti-KIT antibodies of the invention comprise altered glycosylation of amino acid residues. In another embodiment, the altered glycosylation of the amino acid residues results in lowered effector function. In another embodiment, the altered glycosylation of the amino acid residues results in increased effector function. In a specific embodiment, the Fc region has reduced fucosylation. In another embodiment, the Fc region is afucosylated (see for examples, U.S. Patent Application Publication No.

2005/0226867). In one aspect, these antibodies with increased effector function, specifically ADCC, as generated in host cells (e.g., CHO cells, Lemna minor) engineered to produce highly defucosylated antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (Mori et al., 2004, Biotechnol Bioeng 88:901-908; Cox et al., 2006, Nat Biotechnol., 24: 1591-7).

Addition of sialic acid to the oligosaccharides on IgG molecules can enhance their anti-inflammatory activity and alters their cytotoxicity (Keneko et al., Science, 2006, 313:670-673; Scallon et al, Mol. Immuno. 2007 Mar;44(7): 1524-34). The studies referenced above demonstrate that IgG molecules with increased sialylation have anti-inflammatory properties whereas IgG molecules with reduced sialylation have increased

immuno stimulatory properties (e.g., increase ADCC activity). Therefore, an antibody can be modified with an appropriate sialylation profile for a particular therapeutic application (US Publication No. 2009/0004179 and International Publication No. WO 2007/005786).

In one embodiment, the Fc regions of antibodies of the invention comprise an altered sialylation profile compared to the native Fc region. In one embodiment, the Fc regions of antibodies of the invention comprise an increased sialylation profile compared to the native Fc region. In another embodiment, the Fc regions of antibodies of the invention comprise a decreased sialylation profile compared to the native Fc region.

In one embodiment, the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Ghetie et al., 1997, Nat Biotech. 15:637- 40; Duncan et al, 1988, Nature 332:563-564; Lund et al, 1991, J. Immunol 147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation 57: 1537-1543; Hutchins et al, 1995, Proc Natl. Acad Sci U S A 92: 11980-11984; Jefferis et al, 1995, Immunol Lett. 44: 111-117; Lund et al., 1995, Faseb J 9: 115-119; Jefferis et al, 1996, Immunol Lett 54: 101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164: 1925-1933; Xu et al., 2000, Cell Immunol 200: 16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al, 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al, 2002, Biochem Soc Trans 30:487-490); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046;

6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 7,122,637; 7,183,387; 7,332,581; 7,335,742; 7,371,826; 6,821,505; 6,180,377; 7,317,091; 7,355,008;

2004/0002587; and WO 99/58572. Other modifications and/or substitutions and/or additions and/or deletions of the Fc domain will be readily apparent to one skilled in the art.

13. Glycosylation

In addition to the ability of glycosylation to alter the effector function of antibodies, modified glycosylation in the variable region can alter the affinity of the antibody for a target antigen. In one embodiment, the glycosylation pattern in the variable region of the present antibodies is modified. For example, an aglycoslated antibody can be made {i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions can also be made that result in elimination of a glycosylation site present in the Fc region {e.g., Asparagine 297 of IgG). Furthermore, aglycosylated antibodies may be produced in bacterial cells which lack the necessary glycosylation machinery.

14. Antibody Conjugates

The antibodies of the invention may be conjugated or covalently attached to a substance using methods well known in the art. In one embodiment, the attached substance is a therapeutic agent, a detectable label (also referred to herein as a reporter molecule) or a solid support. Suitable substances for attachment to antibodies include, but are not limited to, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, a fluorophore, a chromophore, a dye, a toxin, a hapten, an enzyme, an antibody, an antibody fragment, a radioisotope, solid matrixes, semi-solid matrixes and combinations thereof. In a specific embodiment, the attached substance is a toxin. In another embodiment, the attached substance is an anti-cancer agent. Methods for conjugation or covalently attaching another substance to an antibody are well known in the art.

The antibodies of the invention may also be conjugated to a solid support. Antibodies may be conjugated to a solid support as part of the screening and/or purification and/or manufacturing process. Alternatively antibodies of the invention may be conjugated to a solid support as part of a diagnostic method or composition. A solid support suitable for use in the present invention is typically substantially insoluble in liquid phases. A large number of supports are available and are known to one of ordinary skill in the art. Thus, solid supports include solid and semi-solid matrixes, such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports. More specific examples of solid supports include silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex bead, magnetic bead, paramagnetic bead,

superparamagnetic bead, starch and the like.

In some embodiments, the solid support may include a reactive functional group, including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc., for attaching the antibodies of the invention.

A suitable solid phase support can be selected on the basis of desired end use and suitability for various synthetic protocols. For example, where amide bond formation is desirable to attach the antibodies of the invention to the solid support, resins generally useful in peptide synthesis may be employed, such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE™ resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TENTAGEL™, Rapp Polymere, Tubingen, Germany), polydimethyl-acrylamide resin (available from Milligen/Biosearch, California), or PEGA beads (obtained from Polymer Laboratories).

The antibodies of the invention may also be conjugated to labels for purposes of diagnostics and other assays wherein the antibody and/or its associated ligand may be detected. A label conjugated to an antibody and used in the present methods and

compositions described herein, is any chemical moiety, organic or inorganic, that exhibits an absorption maximum at wavelengths greater than 280 nm, and retains its spectral properties when covalently attached to an antibody. Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.

In certain embodiments, the anti-KIT antibodies are conjugated to a fluorophore. As such, fluorophores used to label antibodies of the invention include, without limitation; a pyrene (including any of the corresponding derivative compounds disclosed in US Patent 5,132,432), an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-l, 3- diazole (NBD), a cyanine (including any corresponding compounds in US Patent

Nos.6,977, 305 and 6,974,873), a carbocyanine (including any corresponding compounds in US Serial Nos. 09/557,275; U.S.; Patents Nos. 4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 Al), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed in US Patent Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), a xanthene (including any corresponding compounds disclosed in U.S. Patent No. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343; 5,227,487;

5,442,045; 5,798,276; 5,846,737; 4,945,171; US serial Nos. 09/129,015 and 09/922,333), an oxazine (including any corresponding compounds disclosed in US Patent No. 4,714,763) or a benzoxazine, a carbazine (including any corresponding compounds disclosed in US Patent No. 4,810,636), a phenalenone, a coumarin (including an corresponding compounds disclosed in US Patent Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (including an corresponding compounds disclosed in US Patent Nos. 4,603,209 and

4,849,362) and benzphenalenone (including any corresponding compounds disclosed in US Patent No. 4,812,409) and derivatives thereof. As used herein, oxazines include resorufins (including any corresponding compounds disclosed in 5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

In a specific embodiment, the fluorophores conjugated to the antibodies described herein include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene. In other embodiments, such fluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines. Also included are dyes sold under the tradenames, and generally known as, Alexa Fluor, DyLight, Cy Dyes, BODIPY, Oregon Green, Pacific Blue, IRDyes, FAM, FITC, and ROX.

The choice of the fluorophore attached to the anti-KIT antibody will determine the absorption and fluorescence emission properties of the conjugated antibody. Physical properties of a fluorophore label that can be used for antibody and antibody bound ligands include, but are not limited to, spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate, or combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, and thereby allow for multiplexed analysis. In certain embodiments, the fluorophore has an absorption maximum at wavelengths greater than 480 nm. In other embodiments, the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp). In other embodiment a fluorophore can emit in the NIR (near infra red region) for tissue or whole organism applications. Other desirable properties of the fluorescent label may include cell permeability and low toxicity, for example if labeling of the antibody is to be performed in a cell or an organism (e.g., a living animal).

In certain embodiments, an enzyme is a label and is conjugated to an anti-KIT antibody. Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity. The enzyme itself does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal. Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal. The enzyme substrate is selected to yield the preferred measurable product, e.g., colorimetric, fluorescent or chemiluminescence. Such substrates are extensively used in the art and are well known by one skilled in the art. In one embodiment, colorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3'- diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively). Other colorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS), ophenylenediamine (OPD), 3,3' ,5,5'-tetramethylbenzidine (TMB), odianisidine, 5-aminosalicylic acid, 4-chloro- l-naphthol. Fluorogenic substrates include, but are not limited to, homo vanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including AMPLEX^® Red reagent and its variants (U.S. Pat. No. 4,384,042) and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat. No. 6,162,931) and dihydrorhodamines including dihydrorhodamine 123. Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are "fixed in place" by the action of a peroxidase in the process described as tyramide signal amplification (TSA). These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by

microscopy, flow cytometry, optical scanning and fluorometry.

In another embodiment, a colorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3- indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o- nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6, 8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat. No. 5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, 9H-(l,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos. 5,316,906 and 5,443,986).

Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta- glucosidase, are additional suitable enzymes. Appropriate colorimetric substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, onitrophenyl beta-D- galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside. In one

embodiment, fluorogenic substrates include resorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl beta-D- galactopyranoside, carboxyumbelliferyl beta-D-galactopyranoside and fluorinated coumarin beta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

Enzymes and their appropriate substrates that produce chemiluminescence are preferred for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful.

In another embodiment, haptens such as biotin, are also utilized as labels. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin- HRP. Subsequently a peroxidase substrate is added to produce a detectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

In certain embodiments, fluorescent proteins may be conjugated to the antibodies as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof. The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents. These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra. This is particularly advantageous for detecting a low quantity of a target in a sample wherein the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein. For this to work, the fluorescent protein and fluorophore function as an energy transfer pair wherein the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorophore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein. A particularly useful combination is the phycobiliproteins disclosed in US Patent Nos.

4,520,110; 4,859,582; 5,055,556 and the sulforhodamine fluorophores disclosed in US Patent No. 5,798,276, or the sulfonated cyanine fluorophores disclosed in US Patent Nos. 6,977,305 and 6,974,873; or the sulfonated xanthene derivatives disclosed in US Patent No. 6,130,101 and those combinations disclosed in US Patent No. 4,542,104. Alternatively, the fluorophore functions as the energy donor and the fluorescent protein is the energy acceptor.

In certain embodiments, the label is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur (³⁵S), tritium (³H), indium (^{Π 1}1η,' ¹¹²In, ¹¹³mln, ¹¹⁵mln,), technetium (⁹⁹Tc, ⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³⁵Xe), fluorine (¹⁸F), ¹⁵³SM, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re,¹⁴²Pr, ¹⁰⁵

III. Screening Assays for Identifying and Characterizing Anti-KIT Antibodies

The antibodies, and antigen-binding portions thereof, of the invention may be screened for KIT inhibitory activity using any of the assays described herein and those assays that are well known in the art. For example, assays which may determine receptor internalization, receptor autophosphorylation, and/or kinase signaling may be used to identify an antibody, or antigen-binding portion thereof, which prevents the activation of the KIT receptor. Screening for new inhibitor antibodies may be accomplished by using standard methods known in the art, for example, by employing a phosphoELISA™ procedure

(available at Invitrogen) to determine the phosphorylation state of KIT or a downstream molecule. The phosphorylation state of the KIT receptor may be determined using commercially available kits such as, for example, C-KIT [pY823] ELISA KIT, HU

(BioSource™; Catalog Number - KHO0401); c-KIT [TOTAL] ELISA KIT, HU

(BioSource™; Catalog Number - KHO0391). Antibodies of the invention may be screened using such kits to determine their KIT inhibitory activity. For example, after treatment with an appropriate ligand and an antibody, or antigen-binding portion thereof, of the invention, a phosphoELISA™ may be performed to determine the phosphorylation state and, thus, the activation state of KIT. Antibodies, or antigen-binding portions thereof, of the invention could be identified as those which prevent KIT activation. The Examples, below, describe assays which involve the detection of KIT activation using based on phosphorylation status. Since receptor activation may lead to endocytosis and receptor internalization, it is useful, in some embodiments, to determine the ability of antibodies, or antigen-binding portions thereof, of the invention to inhibit KIT by measuring their ability to prevent receptor internalization. Receptor internalization assays are well known in the art and described in, for example, Fukunaga et al. (2006) Life Sciences. 80(1). p. 17-23; Bernhagen et al. (2007) Nature Medicine 13, 587 - 596;

natureprotocols. com/2007/04/18/receptor_internalization_assay.php),the entire contents of each of which are incorporated herein by reference. One well-known method to determine receptor internalization is to tag a ligand with a flurorecent protein, e.g., Green Flurorescent Protein (GFP), or other suitable labeling agent. Upon binding of the ligand to the receptor, flurorescence microscopy may be used to visualize receptor internalization. Similarly, an antibody, or antigen-binding portion thereof, of the invention may be tagged with a labeling agent and flurorescence microscopy may be used to visualize ΚΓΓ receptor internalization. If the antibody is able to inhibit the activity of the KIT receptor, lessened internalization of flurorescence in the presence of ligand as compared to appropriate controls {e.g.,

fluorescence may be observed only at the periphery of the cell where the moity binds the KIT receptor rather than in endosomes or vesicles).

In addition to those mentioned above, various other KIT receptor activation assays are known in the art, any of which may be used to evaluate the function of the antibodies of the invention. Further receptor activation assays which may be used in accordance with the present invention are described in U.S. Patent Nos. 6,287,784; 6,025,145; 5,599,681;

5,766,863; 5,891,650; 5,914,237; 7,056,685; and many scientific publications including, but not limited to: Amir-Zaltsman et al. (2000) Luminescence 15(6):377-80; Nakayama and Parandoosh (1999) Journal of Immunological Methods . 225(1-2), 27, 67-74; Pike et al.

(1987) Methods of Enzymology 146: 353-362; Atienza et al. (2005) Journal of Biomolecular Screening. 11(6): 634-643; Hunter et al. (1982). Journal of Biological Chemistry 257(9): 4843-4848; White and Backer (1991) Methods in Enzymology 201: 65-67; Madden et al. (1991) Anal Biochem 199: 210-215; Cleaveland et al. (1990) Analytical Biochemistry 190: 249-253; Lazaro et al. (1991) Analytical Biochemistry 192: 257-261; Hunter and Cooper (1985) Am Rev Biochem 54: 897-930; Ullrich and Schlessinger (1990) Cell 61: 203-212; Knutson and Buck (1991) Archives of Biochemistry and Biophysics 285(2): 197-204); King et al. (1993; Life Sciences 53: 1465-1472; Wang. (1985) Molecular and Cellular Biology 5(12): 3640-3643; Glenney et al. (1988 J Journal of Immunological Methods 109: 277-285; Kamps (1991) Methods in Enzymology 201: 101-110; Kozma et al. (1991 J Methods in Enzymology 201: 28-43; Holmes et al. (1992) Science 256: 1205-10; and Corfas et al. (1993J PNAS, USA 90: 1624-1628.

KIT receptor activation by ligand binding typically initiates subsequent intracellular events, e.g., increases in secondary messengers such as IP₃ which, in turn, releases intracellular stores of calcium ions. Thus, receptor activity may be determined by measuring the quantity of secondary messengers such as IP₃ cyclic nucleotides, intracellular calcium, or phosphorylated signaling molecules such as STAT, PI3K,Grb2, or other possible targets known in the art. U.S. Patent No. 7,056,685 describes and references several methods which may be used in accordance with the present invention to detect receptor activity and is incorporated herein by reference.

Many of the assays described above, such as receptor internalization assays or receptor activation assays may involve the detection or quantification of KIT using immunological binding assays (e.g., when using a radiolabeled antibody to detecting the amount of KIT on the cell surface during a receptor internalization assay). Immunological binding assays are widely described in the art (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).

Immunoassays such as may be employed in receptor internalization studies, receptor activation studies, or receptor detection assays often use a labeling agent to specifically bind to and label the complex formed by the detecting antibody and KIT (see U.S. Pat. No.

7,056,685 which is incorporated herein by reference). The labeling agent may itself be the antibody used to detect the KIT receptor (the antibody here may or may not be an antibodyof the invention). Alternatively, the labeling agent may be a third agent, such as a secondary or tertiary antibody (e.g., and anti-mouse antibody binding to mouse monoclonal antibody specific for KIT). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the labeling agent in an immunological binding assay. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al. (1973), /. Immunol. 111: 1401-1406; Akerstrom et al. (1985), /. Immunol. 135:2589 2542). The labeling agent can also be modified with a detectable agent, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.

Commonly used assays include noncompetitive assays, e.g., sandwich assays, and competitive assays. Commonly used assay formats include Western blots (immunoblots), which are used to detect and quantify the presence of protein in a sample. The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the immunoglobulin used to detect KIT or an antibody of the invention which is designed to bind and inactivate KIT. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like),

3 125 35 14 32

radiolabels (e.g., H, I, S, C, or P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene or latex).

The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. The label can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems that may be used, see U.S. Pat. No. 4,391,904.

Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

In a further aspect of the invention, the antibodies of the present invention may bind to epitopes on KIT and still allow the ectodomain of KIT to dimerize. In this embodiment, the binding of the antibody, or antigen-binding portion thereof, may affect the positioning, orientation and/or distance between the Ig-like domains of the two monomers (e.g., the D4- D4 or D5-D5 domains of KIT), thereby inhibiting the activity of KIT receptor. In other words, the antibody may allow ligand induced dimerization of the KIT ectodomains, but affect the positioning of the two ectodomains at the cell surface interface or alter or prevent conformational changes in KIT, thereby inhibiting the activity of KIT (e.g., inhibiting receptor internalization and/or inhibiting tyrosine autophosphorylation of the receptor and/or inhibiting the ability of the KIT receptor to activate a downstream signaling pathway). Thus, in some embodiments, it is useful to employ assays which are able to identify antibodies that allow KIT receptor dimerization, yet render the KIT receptor inactive. Such assays are described herein.

The conformational state of the KIT receptor may also be determined by Fluorescence Resonance Energy Transfer (FRET) analysis. A comprehensive review of fluorescence methodologies for determining protein conformations and interactions can be found in Johnson (2005) Traffic. 2005 Dec;6(12): 1078-92 which is incorporated herein by reference. In the FRET assay KIT is labeled with appropriate FRET fluorophores. After KIT is labeled, cells expressing the labeled KIT are incubated with test antibodies, or antigen-binding portions thereof, of the invention and the ligand of KIT (e.g., SCF). FRET analysis will allow the observation of conformational changes in KITassociated with ligand binding, KIT dimerization, and/or KIT receptor activation. By this method one of skill in the art may directly assess a protein conformational change which indicates KIT dimerization without downstream activation. There are a number of methods available to perform FRET analysis, and a large portion of the variation arises from the use of different fluorophores or different techniques to incorporate those fluorophores into proteins of interest. FRET fluorophores and analysis methods are well known in the art, and a brief review of FRET technology is available in Heyduk. (2002) Current Opinion in Biotechnology. 13(4). 292-296 and references therein. The following publications expand on the FRET method and are incorporated herein by reference: Kajihara et al. (2006) Nat Methods. 3(ll):923-9; Biener- Ramanujan et al. (2006) Growth Horm IGF Res. l6(4):247-57; Taniguchi et al. (2007 J Biochemistry. 46(18):5349-57; U.S. Patent Nos. 6,689,574; 5,891,646; and WIPO Publication No. WO02/033102. FRET fluorophores may be incorporated into any domain or hinge region of KIT to detect conformational changes {e.g., the D4 or D5 domains of aKIT) provided that the fluorophores do not interfere with the function of KIT or the ability of antibodies of the invention to bind KIT.

Fluorophores useful for FRET are often the same as those useful for Bioluminescence Resonance Energy Transfer (BRET) as discussed below. The most popular FRET method is to engineer reactive cystein residues into a protein of interest. Fluorophores can then easily react with the chosen cystein residies. Often fusion proteins are constructed, whereby a protein of interest is fused to Green Fluorescent Protein (see Neininger et al. (2001) EMBO Reports. 2(8):703-708). Additional methods and useful fluorophores for FRET are described in Huebsch and Mooney (2007) Biomaterials. 28(15):2424-37; Schmid and Birbach (2007) Thromb Haemost. 97(3):378-84; Jares-Erijman AND Jovin (2006) Curr Opin Chem Biol. 10(5):409-16; Johansson (2006) Methods Mol Biol. 335: 17-29; Wallrabe and Periasamy (2005) Curr Opin Biotechnol. 16(1): 19-27; and Clegg RM (1995) Curr Opin Biotechnol. 6(1): 103-10 which are incorporated herein by reference.

In other embodiments, it may be unknown or difficult to determine which KIT conformation is specifically indicative of dimerization without activation. In such cases, one of skill in the art may combine assays that determine receptor dimerization with those that determine receptor activation. For example, one may use traditional cross-linking studies (exemplified by Rodriguez et al. (1990) Molecular Endocrinology, 4(12), 1782-1790) to detect KIT receptor dimerization in combination with any of the receptor activation assays discussed above. FRET and similar systems may also be used to directly measure receptor activation or dimerization. For example, by incorporating appropriate FRET fluorophores into the cytoplasmic domain of KIT and into a phosphorylation target protein {i.e., a downstream signaling molecule), FRET would be capable of determining whether

downstream signaling molecules were being recruited to KIT. Therefore, in one embodiment a successful antibody, or antigen-binding portion thereof, of the invention is one which allows receptor dimerization, as measured by cross-linking or FRET, but which prevents KIT receptor activation, detected as lack of fluorescence by FRET or BRET analysis or by other receptor activation assays {e.g., autophosphorylation assay employing anti-phosphotyrosine antibodies and Western Blot). Thus, using the techniques described herein, one of skill in the art can easily test antibodies, or antigen-binding portions thereof, to determine whether they inhibit ΚΓΓ receptor activity and whether they allow KIT receptor dimerization.

In particular, Bioluminescence Resonance Energy Transfer (BRET) analysis may be used to identify antibodies which inhibit the activity of KIT. U.S. Pat. Pub. No.

20060199226, WIPO Publication No. WO06/094073, and Tan et al. (2007. Molecular Pharmacology. 72: 1440-1446) specifically describe methods to identify ligands which activate receptor tyrosine kinases and are thus incorporated herein by reference. These techniques have been employed for determining protein interactions in vitro and in vivo (Pfleger et al. (2006) Nature Protocols 1 337-345; Kroeger et al. (2001), J. Biol. Chem., 276(16): 12736-43; and Harikumar, et al. (2004) Mol Pharmacol 65:28-35; which are all incorporated herein by reference). BRET is useful for identifying antibodies of the present invention from test compounds by screening for those antibodies which prevent KIT activation.

As discussed in U.S. Pat. Publication No. 2006/0199226 which is incorporated herein by reference, BRET based assays can be used to monitor the interaction of proteins having a bioluminescent donor molecule (DM) with proteins having a fluorescent acceptor moiety (AM). Briefly, cells expressing a KIT-DM fusion will convert the substrate's chemical energy into light. If there is an AM (e.g., a signaling protein- AM fusion) in close proximity to the KIT-DM fusion, then the cells will emit light at a certain wavelength. For example, BRET based assays can be used to assess the interaction between a KIT-luciferase fusion and a GFP- signalling protein fusion. This differs slightly from FRET analysis, where the donor molecule may be excited by light of a specific wavelength rather than by chemical energy conversion. Examples of bioluminescent proteins with luciferase activity that may be used in a BRET analysis may be found in U.S. Pat. Nos. 5,229,285, 5,219,737, 5,843,746, 5,196,524, 5,670,356. Alternative DMs include enzymes, which can act on suitable substrates to generate a luminescent signal. Specific examples of such enzymes are beta-galactosidase, alkaline phosphatase, beta-glucuronidase and beta-glucosidase. Synthetic luminescent substrates for these enzymes are well known in the art and are commercially available from companies, such as Tropix Inc. (Bedford, Mass., USA). DMs can also be isolated or engineered from insects (U.S. Pat. No. 5,670,356).

Depending on the substrate, DMs emit light at different wavelengths. Non-limiting examples of substrates for DMs include coelenterazine, benzothiazole, luciferin, enol formate, terpene, and aldehyde, and the like. The DM moiety can be fused to either the amino terminal or carboxyl terminal portion of the KIT protein. Preferably, the positioning of the BDM domain within the KIT-DM fusion does not alter the activity of the native protein or the binding of antibodies of the present invention. KIT-DM fusion proteins can be tested to ensure that it retains biochemical properties, such as ligand binding and ability to interact with downstream signaling molecules of the native protein.

AMs in BRET analysis may re-emit the transferred energy as fluorescence. Examples of AMs include Green Fluorescent Protein (GFP), or isoforms and derivatives thereof such as YFP, EGFP, EYFP and the like (R. Y. Tsien, (1998) Ann. Rev. Biochem. 63:509-544).

Preferably, the positioning of the AM domain within the AM- protein fusion does not alter the activity of the native protein. AM-second protein fusion proteins can be tested to ensure that it retains biochemical properties of the cognate native protein, such as interaction with KIT. By way of example, an amino terminal fusion of the GFP protein to any substrate which is phosphorylated by or can bind to ΚΓΓ can be used.

IV. Methods of Using Anti-KIT Antibodies

1. Diagnostic Methods of Use

The anti-KIT antibodies of the invention may be used in vivo and/or in vitro for detecting KIT expression in cells and tissue or for imaging KIT expressing cells and tissues. The diagnostic methods of the invention are suitable for detecting KIT, a fragment thereof or a mutant or variant thereof, e.g., a naturally occurring mutant responsible for causing a disease, such as a KIT associated disease, e.g., cancer, e.g., GIST, AML or SCLC. In certain embodiments, the antibodies are human antibodies and such antibodies are used to image KIT expression in a living human patient. Moreover, given that exemplary antibodies of the invention specifically bind to human KIT, as well as KIT from one or more of mouse, rat, or cynomolgous, such antibodies are useful for in vitro or in vivo imaging, testing, or analysis of samples in animal models.

By way of example, diagnostic uses can be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the antibody under conditions that allow for formation of a complex between the antibody and KIT, or a fragment or mutant thereof. Complex formation is then detected (e.g., using an ELISA or by imaging to detect a moiety attached to the antibody). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of KIT, or a fragment or mutant thereof, in the test sample.

In one embodiment, the invention provides a method of determining the presence of KIT in a sample suspected of containing KIT, by exposing the sample to an anti-KIT antibody of the invention, and determining binding of the antibody to KIT in the sample wherein binding of the antibody to KIT in the sample is indicative of the presence of the KIT in the sample. In one embodiment, the sample is a biological sample.

In certain embodiments, the anti-KIT antibodies may be used to detect the

overexpression or amplification of KIT using an in vivo or ex vivo diagnostic assay. In one embodiment, the anti-KIT antibody is added to a sample wherein the antibody binds the KIT to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label. In one embodiment, the sample is a biological sample. The biological sample may be from a mammal

experiencing or suspected of experiencing a KIT associated disease/disorder.

Alternatively, or additionally, FISH assays such as the INFORM™ (sold by Ventana, Ariz.) or PATHVISION™ (Vysis, 111.) may be carried out on formalin-fixed, paraffin- embedded tissue to determine the extent (if any) of KIT expression or overexpression in a sample.

In certain embodiments, the anti-KIT antibodies and compositions thereof of the invention may be used in vivo and/or in vitro for diagnosing KIT associated diseases. This can be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the antibody under conditions that allow for formation of a complex between the antibody and KIT. Complex formation is then detected (e.g., using an ELISA). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of KIT in the test sample.

In certain embodiments, the anti-KIT antibodies may be used in a method of detecting soluble KIT in blood or serum. In one embodiment, the method comprises contacting a test sample of blood or serum from a mammal suspected of experiencing a KIT disorder with an anti-KIT antibody of the invention and detecting an increase in soluble KIT in the test sample relative to a control sample of blood or serum, e.g., a sample from a non-diseased mammal. In one embodiment, the method of detecting is useful as a method of diagnosing a KIT disorder associated with an increase in soluble KIT in blood or serum of a mammal. 2. Therapeutic Methods of Use

The present invention further provides methods for treating a KIT-associated disease in a subject. The methods include administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, of the invention. The anti-KIT antibodies, or antigen-binding portions thereof, of the present invention have numerous in vitro and in vivo therapeutic utilities involving the treatment of a KIT-associated disease. The antibodies of the present invention can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat or prevent a KIT-associated disease. For example, the human monoclonal antibodies, the multispecific or bispecific molecules, can be used to elicit in vivo or in vitro one or more of the following biological activities: to inhibit the growth of and/or kill a cell expressing KIT; to mediate phagocytosis or ADCC of a cell expressing KIT in the presence of human effector cells; or to lock the ectodomain of KIT to an inactive state and/or a monomeric state thereby antagonizing the activity of KIT.

As used herein "a KIT-associated disease" is a disease or condition which is mediated by KIT activity or is associated with aberrant KIT expression or activation, such as age- related macular degeneration (AMD), atherosclerosis, rheumatoid arthritis, diabetic retinopathy or pain associated diseases. Specific examples of KIT-associated diseases include, but are not limited to, gastrointestinal stromal tumors (GIST), acute myelogenous leukemia (AML), small cell lung cancer (SCLC), breast cancer, bone metastatic breast cancer and tenosynovial giant cell tumors. Additional examples of KIT-associated diseases include colon cancer (including small intestine cancer), lung cancer, breast cancer, pancreatic cancer, melanoma (e.g., metastatic malignant melanoma), acute myeloid leukemia, kidney cancer, bladder cancer, ovarian cancer and prostate cancer. Examples of other cancers that may be treated using the methods of the invention include renal cancer (e.g., renal cell carcinoma), glioblastoma, brain tumors, chronic or acute leukemias including acute lymphocytic leukemia (ALL), adult T-cell leukemia (T-ALL), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), entroblastic/centrocytic (cb/cc) follicular lymphomas cancers, diffuse large cell lymphomas of B lineage, angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma and HIV associated body cavity based lymphomas), embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke's tumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia and other B-cell lymphomas, nasopharangeal carcinomas, bone cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, e.g., mesothelioma and combinations of said cancers.

As used herein, the term "subject" is intended to include human and non-human animals. Non-human animals includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Preferred subjects include human subjects having a KIT-associated disease.

Suitable routes of administering the anti-KIT antibodies, or antigen-binding portions thereof, of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the anti-KIT antibodies can be administered by injection (e.g., intravenous or subcutaneous), as discussed above. Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.

As previously described, the anti-KIT antibodies of the invention can be coadministered with one or other more therapeutic agents, e.g., a toxin, a cytotoxic agent, a radiotoxic agent, an anti-cancer agent, or an immunosuppressive agent. The antibody can be linked to the agent or can be administered separate from the agent. In the latter case (separate administration), the antibody, or antigen-binding portion thereof, can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin is intravenously administered as a 100 mg/ dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days. Co-administration of the anti-KIT antibodies of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.

When administering anti-KIT antibody conjugates of the present invention for use in the prophylaxis and/or treatment of diseases related to abnormal cellular proliferation, a circulating concentration of administered conjugate of about 0.001 μΜ to 20 μΜ or about 0.01 μΜ to 5 μΜ may be used.

Patient doses for oral administration of the antibody, or antigen-binding portion thereof, described herein, typically range from about 1 mg/day to about 10,000 mg/day, more typically from about 10 mg/day to about 1,000 mg/day, and most typically from about 50 mg/day to about 500 mg/day. Stated in terms of patient body weight, typical dosages range from about 0.01 to about 150 mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and most typically from about 1 to about 10 mg/kg/day, for example 5 mg/kg/day or 3 mg/kg/day.

In at least some embodiments, patient doses that retard or inhibit tumor growth can be 1 μιηοΐ/kg/day or less. For example, the patient doses can be 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or 0.1 μιηοΐ/kg/day or less (referring to moles of the drug). Preferably, the anti-KIT- drug conjugate retards growth of the tumor when administered in the daily dosage amount over a period of at least five days.

In one embodiment, conjugates of the invention can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxoins immunosuppressants, etc.) to cells which have KIT cell surface receptors by linking such compounds to the anti-KIT antibody, or antigen-binding portion thereof. For example, an anti-KIT antibody can be conjugated to any of the toxin compounds described in US Patent Nos. 6,281,354 and 6,548,530, US patent publication Nos. 20030050331, 20030064984, 20030073852 and 20040087497 or published in WO 03/022806, which are hereby incorporated by reference in their entireties. Thus, the invention also provides methods for localizing ex vivo or in vivo cells expressing KIT (e.g., with a detectable label, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme co-factor).

Target- specific effector cells, e.g., effector cells linked to compositions (e.g., antibodies, antigen binding portions thereof, small molecules, or peptidic molecules ) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. The target- specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution. The number of

8 9

cells administered can be in the order of 10 -10" but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., a tumor cell expressing KIT and to effect cell killing by, e.g., phagocytosis. Routes of administration can also vary.

Therapy with target- specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-tumor therapy using an antibody, or antigen-binding portion thereof, of the invention and/or effector cells armed with these compositions can be used in conjunction with chemotherapy.

V. Pharmaceutical Formulations of Anti-KIT Antibodies

The present invention further provides preparations and formulations comprising the anti-KIT antibodies (including antibody fragments) of the present invention. It should be understood that any of the anti-KIT antibodies and antibody fragments described herein, including antibodies and antibody fragments having any one or more of the structural and functional features described in detail throughout the application, may be formulated or prepared as described below. When various formulations are described in this section as including an antibody, it is understood that such an antibody may be an antibody or an antibody fragment having any one or more of the characteristics of the anti-KIT antibodies and antibody fragments described herein.

In certain embodiments, the anti-KIT antibodies of the invention may be formulated with a pharmaceutically acceptable carrier as pharmaceutical (therapeutic) compositions, and may be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The term "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the antibodies of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The formulations of the invention are present in a form known in the art and acceptable for therapeutic uses. In one embodiment, a formulation of the invention is a liquid formulation. In another embodiment, a formulation of the invention is a lyophilized formulation. In a further embodiment, a formulation of the invention is a reconstituted liquid formulation. In one embodiment, a formulation of the invention is a stable liquid

formulation. In one embodiment, a liquid formulation of the invention is an aqueous formulation. In another embodiment, the liquid formulation is non-aqueous. In a specific embodiment, a liquid formulation of the invention is an aqueous formulation wherein the aqueous carrier is distilled water.

The formulations of the invention comprise an anti-KIT antibody in a concentration resulting in a w/v appropriate for a desired dose. The anti-KIT antibody may be present in the formulation at a concentration of about lmg/ml to about 500mg/ml, e.g., at a

concentration of at least 1 mg/ml, at least 100 mg/ml, at least 250 mg/ml, or at least 500 mg/ml.

In one embodiment, the concentration of anti-KIT antibody, which is included in the formulation of the invention, is between about 1 mg/ml and about 250 mg/ml or between about 250 mg/ml and about 500 mg/ml.

The formulations of the invention comprising an anti-KIT antibody may further comprise one or more active compounds as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such additional active compound/s is/are suitably present in combination in amounts that are effective for the purpose intended. The formulations of the invention may be prepared for storage by mixing the anti-KIT antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, including, but not limited to buffering agents, saccharides, salts, surfactants, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelators, preservatives, or the like {Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)), in the form of lyophilized formulations or aqueous solutions at a desired final concentration. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as histidine, phosphate, citrate, glycine, acetate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;

cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including trehalose, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes {e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine, or acetate. The saccharide excipient may be trehalose, sucrose, mannitol, maltose or raffinose. The surfactant may be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KC1, MgCl₂, or CaCl₂

The formulations of the invention may include a buffering or pH adjusting agent to provide improved pH control. A formulation of the invention may have a pH of between about 3.0 and about 9.0, between about 4.0 and about 8.0, between about 5.0 and about 8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0, between about 5.5 and about 7.0, or between about 5.5 and about 6.5. In a further embodiment, a formulation of the invention has a pH of about 3.0, about 3.5, about 4.0, about

4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about

6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, a formulation of the invention has a pH of about 6.0. One of skill in the art understands that the pH of a formulation generally should not be equal to the isoelectric point of the particular anti-KIT antibody to be used in the formulation.

Typically, the buffering agent is a salt prepared from an organic or inorganic acid or base. Representative buffering agents include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. In addition, amino acid components can also function in a buffering capacity. Representative amino acid components which may be utilized in the formulations of the invention as buffering agents include, but are not limited to, glycine and histidine. In certain

embodiments, the buffering agent is chosen from histidine, citrate, phosphate, glycine, and acetate. In a specific embodiment, the buffering agent is histidine. In another specific embodiment, the buffering agent is citrate. In yet another specific embodiment, the buffering agent is glycine. The purity of the buffering agent should be at least 98%, or at least 99%, or at least 99.5%. As used herein, the term "purity" in the context of histidine and glycine refers to chemical purity of histidine or glycine as understood in the art, e.g., as described in The Merck Index, 13^th ed., O'Neil et al. ed. (Merck & Co., 2001).

Buffering agents are typically used at concentrations between about 1 mM and about 200 mM or any range or value therein, depending on the desired ionic strength and the buffering capacity required. The usual concentrations of conventional buffering agents employed in parenteral formulations can be found in: Pharmaceutical Dosage Form:

Parenteral Medications, Volume 1, 2^nd Edition, Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume Parenterals", Table 5: Commonly used additives in Parenteral Products. In one embodiment, the buffering agent is at a concentration of about 1 mM, or of about 5 mM, or of about 10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or of about 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, or of about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM, or of about 90 mM, or of about 100 mM. In one embodiment, the buffering agent is at a concentration of 1 mM, or of 5 mM, or of 10 mM, or of 15 mM, or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, or of 45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90 mM, or of 100 mM. In a specific embodiment, the buffering agent is at a concentration of between about 5 mM and about 50 mM. In another specific embodiment, the buffering agent is at a concentration of between 5 mM and 20 mM. In certain embodiments, the formulation of the invention comprises histidine as a buffering agent. In one embodiment the histidine is present in the formulation of the invention at a concentration of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM histidine. In another embodiment, a formulation of the invention comprises between about 1 mM and about 200 mM, between about 1 mM and about 150 mM, between about 1 mM and about 100 mM, between about 1 mM and about 75 mM, between about 10 mM and about 200 mM, between about 10 mM and about 150 mM, between about 10 mM and about 100 mM, between about 10 mM and about 75 mM, between about 10 mM and about 50 mM, between about 10 mM and about 40 mM, between about 10 mM and about 30 mM, between about 20 mM and about 75 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, or between about 20 mM and about 30 mM histidine. In a further embodiment, the formulation comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In a specific embodiment, a formulation may comprise about 10 mM, about 25 mM, or no histidine.

The formulations of the invention may comprise a carbohydrate excipient.

Carbohydrate excipients can act, e.g., as viscosity enhancing agents, stabilizers, bulking agents, solubilizing agents, and/or the like. Carbohydrate excipients are generally present at between about 1% to about 99% by weight or volume, e.g., between about 0.1% to about 20%, between about 0.1 % to about 15%, between about 0.1% to about 5%, , between about 1% to about 20%, between about 5% to about 15%, between about 8% to about 10%, between about 10% and about 15%, between about 15% and about 20%, between 0.1% to 20%, between 5% to 15%, between 8% to 10%, between 10% and 15%, between 15% and 20%, between about 0.1 % to about 5%, between about 5% to about 10%, or between about 15% to about 20%. In still other specific embodiments, the carbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of the invention include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like. In one embodiment, the carbohydrate excipients for use in the present invention are chosen from, sucrose, trehalose, lactose, mannitol, and raffinose. In a specific embodiment, the carbohydrate excipient is trehalose. In another specific embodiment, the carbohydrate excipient is mannitol. In yet another specific embodiment, the carbohydrate excipient is sucrose. In still another specific embodiment, the carbohydrate excipient is raffinose. The purity of the carbohydrate excipient should be at least 98%, or at least 99%, or at least 99.5%.

In a specific embodiment, the formulations of the invention may comprise trehalose. In one embodiment, a formulation of the invention comprises at least about 1%, at least about 2%, at least about 4%, at least about 8%, at least about 20%, at least about 30%, or at least about 40% trehalose. In another embodiment, a formulation of the invention comprises between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20%, between about 2% and about 40%, between about 2% and about 30%, between about 2% and about 20%, between about 4% and about 40%, between about 4% and about 30%, or between about 4% and about 20% trehalose. In a further embodiment, a formulation of the invention comprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%, about 20%, about 30%, or about 40% trehalose. In a specific embodiment, a formulation of the invention comprises about 4%, about 6% or about 15% trehalose.

In certain embodiments, a formulation of the invention comprises an excipient. In a specific embodiment, a formulation of the invention comprises at least one excipient chosen from: sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier and preservative. In one embodiment, a formulation of the invention comprises a salt, e.g., a salt selected from: NaCl, KC1, CaCl₂, and MgCl₂. In a specific embodiment, the formulation comprises NaCl.

A formulation of the invention may comprise at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, at least about 80 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, or at least about 300 mM sodium chloride (NaCl). In a further embodiment, the formulation may comprise between about 10 mM and about 300 mM, between about 10 mM and about 200 mM, between about 10 mM and about 175 mM, between about 10 mM and about 150 mM, between about 25 mM and about 300 mM, between about 25 mM and about 200 mM, between about 25 mM and about 175 mM, between about 25 mM and about 150 mM, between about 50 mM and about 300 mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 75 mM and about 300 mM, between about 75 mM and about 200 mM, between about 75 mM and about 175 mM, between about 75 mM and about 150 mM, between about 100 mM and about 300 mM, between about 100 mM and about 200 mM, between about 100 mM and about 175 mM, or between about 100 mM and about 150 mM sodium chloride. In a further embodiment, the formulation may comprise about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, or about 300 mM sodium chloride.

A formulation of the invention may also comprise an amino acid, e.g., lysine, arginine, glycine, histidine or an amino acid salt. The formulation may comprise at least about ImM, at least about lOmM, at least about 25 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, or at least about 400 mM of an amino acid. In another embodiment, the formulation may comprise between about 1 mM and about 100 mM, between about 10 mM and about 150 mM, between about 25 mM and about 250 mM, between about 25 mM and about 300 mM, between about 25 mM and about 350 mM, between about 25 mM and about 400 mM, between about 50 mM and about 250 mM, between about 50 mM and about 300 mM, between about 50 mM and about 350 mM, between about 50 mM and about 400 mM, between about 100 mM and about 250 mM, between about 100 mM and about 300 mM, between about 100 mM and about 400 mM, between about 150 mM and about 250 mM, between about 150 mM and about 300 mM, or between about 150 mM and about 400 mM of an amino acid. In a further embodiment, a formulation of the invention comprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of an amino acid.

The formulations of the invention may further comprise a surfactant. The term "surfactant" as used herein refers to organic substances having amphipathic structures;

namely, they are composed of groups of opposing solubility tendencies, typically an oil- soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface- active moiety, into anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting, emulsifying, solubilizing, and dispersing agents for various pharmaceutical compositions and preparations of biological materials. Pharmaceutically acceptable surfactants like polysorbates (e.g., polysorbates 20 or 80);

polyoxamers (e.g., poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.,

lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the

MONAQUA™ series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.), can optionally be added to the formulations of the invention to reduce aggregation. In one embodiment, a formulation of the invention comprises Polysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80. Surfactants are particularly useful if a pump or plastic container is used to administer the formulation. The presence of a pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate. The formulations may comprise a polysorbate which is at a concentration ranging from between about 0.001 to about 1%, or about 0.001% to about 0.1%, or about 0.01% to about 0.1%. In other specific embodiments, the formulations of the invention comprise a polysorbate which is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.

The formulations of the invention may optionally further comprise other common excipients and/or additives including, but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, or the like. Pharmaceutically acceptable excipients and/or additives may be used in the formulations of the invention. Commonly used excipients/additives, such as pharmaceutically acceptable chelators (for example, but not limited to, EDTA, DTPA or EGTA) can optionally be added to the formulations of the invention to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (for example, but not limited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof can optionally be added to the formulations of the invention at any suitable concentration such as between about 0.001% to about 5%, or any range or value therein. The concentration of preservative used in the formulations of the invention is a concentration sufficient to yield a microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in the formulations of the invention include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, salt-forming counterions such as sodium and the like. These and additional known pharmaceutical excipients and/or additives suitable for use in the formulations of the invention are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 21^st ed., Lippincott Williams & Wilkins, (2005), and in the "Physician's Desk Reference", 60^th ed., Medical Economics, Montvale, N.J. (2005). Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of anti-KIT antibody, as well known those in the art or as described herein.

It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood, wherein the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations will generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, using a vapor pressure or ice-freezing type osmometer. Tonicity of a formulation is adjusted by the use of tonicity modifiers. "Tonicity modifiers" are those pharmaceutically acceptable inert substances that can be added to the formulation to provide an isotonity of the formulation. Tonicity modifiers suitable for this invention include, but are not limited to, saccharides, salts and amino acids.

In certain embodiments, the formulations of the present invention have an osmotic pressure from about 100 mOSm to about 1200 mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800 mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or from about 250 mOSm to about 350 mOSm.

The concentration of any one component or any combination of various components, of the formulations of the invention is adjusted to achieve the desired tonicity of the final formulation. For example, the ratio of the carbohydrate excipient to antibody may be adjusted according to methods known in the art (e.g., U.S. Patent No. 6,685,940). In certain embodiments, the molar ratio of the carbohydrate excipient to antibody may be from about 100 moles to about 1000 moles of carbohydrate excipient to about 1 mole of antibody, or from about 200 moles to about 6000 moles of carbohydrate excipient to about 1 mole of antibody, or from about 100 moles to about 510 moles of carbohydrate excipient to about 1 mole of antibody, or from about 100 moles to about 600 moles of carbohydrate excipient to about 1 mole of antibody.

The desired isotonicity of the final formulation may also be achieved by adjusting the salt concentration of the formulations. Pharmaceutically acceptable salts and those suitable for this invention as tonicity modifiers include, but are not limited to, sodium chloride, sodium succinate, sodium sulfate, potassuim chloride, magnesium chloride, magnesium sulfate, and calcium chloride. In specific embodiments, formulations of the invention comprise NaCl, MgCl₂, and/or CaCl₂. In one embodiment, concentration of NaCl is between about 75 mM and about 150 mM. In another embodiment, concentration of MgCl₂ is between about 1 mM and about 100 mM. Pharmaceutically acceptable amino acids including those suitable for this invention as tonicity modifiers include, but are not limited to, proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the invention are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram body weight, as can be the case with antibodies, even trace amounts of harmful and dangerous endotoxin must be removed. In certain specific embodiments, the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. When used for in vivo administration, the formulations of the invention should be sterile. The formulations of the invention may be sterilized by various sterilization methods, including sterile filtration, radiation, etc. In one embodiment, the antibody formulation is filter- sterilized with a presterilized 0.22-micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy", 21^st ed., Lippincott Williams & Wilkins, (2005).

Formulations comprising antibodies, such as those disclosed herein, ordinarily will be stored in lyophilized form or in solution. It is contemplated that sterile compositions comprising antibodies are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle. In one embodiment, a composition of the invention is provided as a pre-filled syringe.

In one embodiment, a formulation of the invention is a lyophilized formulation. The term "lyophilized" or "freeze-dried" includes a state of a substance that has been subjected to a drying procedure such as lyophilization, where at least 50% of moisture has been removed.

The phrase "bulking agent" includes a compound that is pharmaceutically acceptable and that adds bulk to a lyo cake. Bulking agents known to the art include, for example, carbohydrates, including simple sugars such as dextrose, ribose, fructose and the like, alcohol sugars such as mannitol, inositol and sorbitol, disaccharides including trehalose, sucrose and lactose, naturally occurring polymers such as starch, dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin), glycogen, and synthetic monomers and polymers.

A "lyoprotectant" is a molecule which, when combined with a protein of interest (such as an anti-KIT antibody), significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage. Lyoprotectants include, but are not limited to, sugars and their corresponding sugar alcohols; an amino acid such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher molecular weight sugar alcohols, e.g., glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; PLURONICS™; and combinations thereof. Additional examples of lyoprotectants include, but are not limited to, glycerin and gelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose and stachyose. Examples of reducing sugars include, but are not limited to, glucose, maltose, lactose, maltulose, iso-maltulose and lactulose. Examples of non-reducing sugars include, but are not limited to, non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols. Examples of sugar alcohols include, but are not limited to, monoglycosides, compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The glycosidic side group can be either glucosidic or galactosidic. Additional examples of sugar alcohols include, but are not limited to, glucitol, maltitol, lactitol and iso- maltulose. In specific embodiments, trehalose or sucrose is used as a lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a "lyoprotecting amount" which means that, following lyophilization of the protein in the presence of the lyoprotecting amount of the lyoprotectant, the protein essentially retains its physical and chemical stability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose) and anti-KIT antibody molecules of a formulation of the invention is at least about 10, at least about 50, at least about 100, at least about 200, or at least about 300. In another embodiment, the molar ratio of a lyoprotectant (e.g., trehalose) and anti-KIT antibody molecules of a formulation of the invention is about 1, is about 2, is about 5, is about 10, about 50, about 100, about 200, or about 300.

A "reconstituted" formulation is one which has been prepared by dissolving a lyophilized antibody formulation in a diluent such that the antibody is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient to be treated with the anti-KIT antibody and, in certain embodiments of the invention, may be one which is suitable for intravenous administration.

The "diluent" of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization. In some embodiments, diluents include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution. In an alternative embodiment, diluents can include aqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the invention is a lyophilized formulation comprising an anti-KIT antibody of the invention, wherein at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of said antibody may be recovered from a vial upon shaking said vial for 4 hours at a speed of 400 shakes per minute wherein the vial is filled to half of its volume with the formulation. In another embodiment, a formulation of the invention is a lyophilized formulation comprising an anti-KIT antibody of the invention, wherein at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the antibody may be recovered from a vial upon subjecting the formulation to three freeze/thaw cycles wherein the vial is filled to half of its volume with said formulation. In a further embodiment, a formulation of the invention is a lyophilized formulation comprising an anti-KIT antibody of the invention, wherein at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the antibody may be recovered by reconstituting a lyophilized cake generated from said formulation.

In one embodiment, a reconstituted liquid formulation may comprise an anti-KIT antibody at the same concentration as the pre-lyophilized liquid formulation.

In another embodiment, a reconstituted liquid formulation may comprise an anti-KIT antibody at a higher concentration than the pre-lyophilized liquid formulation, e.g., .about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold higher concentration of an anti-KIT antibody than the pre-lyophilized liquid formulation.

In yet another embodiment, a reconstituted liquid formulation may comprise an anti- KIT antibody of the invention at a lower concentration than the pre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold or about 10 fold lower concentration of an anti-KIT antibody than the pre-lyophilized liquid formulation.

The pharmaceutical formulations of the invention are preferably stable formulations, e.g., stable at room temperature.

The terms "stability" and "stable" as used herein in the context of a formulation comprising an anti-KIT antibody of the invention refer to the resistance of the antibody in the formulation to aggregation, degradation or fragmentation under given manufacture, preparation, transportation and storage conditions. The "stable" formulations of the invention retain biological activity under given manufacture, preparation, transportation and storage conditions. The stability of the anti-KIT antibody can be assessed by degrees of aggregation, degradation or fragmentation, as measured by HPSEC, static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), urea unfolding techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and/or ANS binding techniques, compared to a reference formulation. For example, a reference formulation may be a reference standard frozen at -70°C consisting of 10 mg/ml of an anti- KIT antibody of the invention in PBS.

Therapeutic formulations of the present invention may be formulated for a particular dosage. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the anti-KIT antibody and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an anti-KIT antibody for the treatment of sensitivity in individuals.

Therapeutic compositions of the present invention can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. By way of example, in certain embodiments, the antibodies (including antibody fragments) are formulated for intravenous administration. In certain other embodiments, the antibodies (including antibody fragments) are formulated for local delivery to the cardiovascular system, for example, via catheter, stent, wire, intramyocardial delivery, intrapericardial delivery, or intraendocardial delivery.

Formulations of the present invention which are suitable for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required (US Patent No. 7,378,110; 7,258,873; 7,135,180; US Publication No. 2004- 0042972; and 2004-0042971).

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

In certain embodiments, antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention can cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;

5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett.

357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the invention, as well as components of the invented molecules; pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L.

Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in another embodiment, the liposomes include a targeting moiety. In another embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area. When administered in this manner, the composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. Additionally or alternatively, the antibodies of the invention may be delivered locally to the brain to mitigate the risk that the blood brain barrier slows effective delivery.

In certain embodiments, the therapeutic antibody compositions may be administered with medical devices known in the art. For example, in certain embodiments an antibody or antibody fragment is administered locally via a catheter, stent, wire, or the like. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos.

5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

The efficient dosages and the dosage regimens for the antibodies of the invention depend on the disease or condition to be treated and can be determined by the persons skilled in the art.

A "therapeutically effective dosage" for treating a KIT-associated disease may, for example, decrease tumor size, decrease tumor metastasis, decrease tumor vascularization, etc. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

VI. Kits and Articles of Manufacture Comprising Anti-KIT Antibodies

Also within the scope of the present invention are kits comprising the anti-KIT antibodies, and antigen-binding portions thereof, and instructions for use. The term "kit" as used herein refers to a packaged product comprising components with which to administer the anti-KIT antibody, or antigen-binding portion thereof, of the invention for treatment of a KIT-related disorder. The kit preferably comprises a box or container that holds the components of the kit. The box or container is affixed with a label or a Food and Drug Administration approved protocol. The box or container holds components of the invention which are preferably contained within plastic, polyethylene, polypropylene, ethylene, or propylene vessels. The vessels can be capped-tubes or bottles. The kit can also include instructions for administering an anti-KIT antibody.

The kit can further contain one more additional reagents, such as an

immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent or one or more additional anti-KIT antibodies of the invention (e.g., an anti-KIT antibody having a complementary activity which binds to an epitope in the KIT antigen distinct from the first anti-KIT antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with a liquid formulation or lyophilized formulation of an anti-KIT antibody or antibody fragment thereof. In one embodiment, a container filled with a liquid formulation of the invention is a pre-filled syringe. In a specific embodiment, the formulations of the invention are formulated in single dose vials as a sterile liquid. For example, the

formulations may be supplied in 3 cc USP Type I borosilicate amber vials (West

Pharmaceutical Services - Part No. 6800-0675) with a target volume of 1.2 mL. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In one embodiment, a container filled with a liquid formulation of the invention is a pre-filled syringe. Any pre-filled syringe known to one of skill in the art may be used in combination with a liquid formulation of the invention. Pre-filled syringes that may be used are described in, for example, but not limited to, PCT Publications WO05032627,

WO08094984, W09945985, WO03077976, US Patents US6792743, US5607400,

US5893842, US7081107, US7041087, US5989227, US6807797, US6142976, US5899889, US Patent Publications US20070161961A1, US20050075611A1, US20070092487A1, US20040267194A1, US20060129108A1. Pre-filled syringes may be made of various materials. In one embodiment a pre-filled syringe is a glass syringe. In another embodiment a pre-filled syringe is a plastic syringe. One of skill in the art understands that the nature and/or quality of the materials used for manufacturing the syringe may influence the stability of a protein formulation stored in the syringe. For example, it is understood that silicon based lubricants deposited on the inside surface of the syringe chamber may affect particle formation in the protein formulation. In one embodiment, a pre-filled syringe comprises a silicone based lubricant. In one embodiment, a pre-filled syringe comprises baked on silicone. In another embodiment, a pre-filled syringe is free from silicone based lubricants. One of skill in the art also understands that small amounts of contaminating elements leaching into the formulation from the syringe barrel, syringe tip cap, plunger or stopper may also influence stability of the formulation. For example, it is understood that tungsten introduced during the manufacturing process may adversely affect formulation stability. In one embodiment, a pre-filled syringe may comprise tungsten at a level above 500 ppb. In another embodiment, a pre-filled syringe is a low tungsten syringe. In another embodiment, a pre-filled syringe may comprise tungsten at a level between about 500 ppb and about 10 ppb, between about 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb, between about 100 ppb and about 10 ppb, between about 50 ppb and about 10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising anti-KIT antibodies are also provided that are useful for various purposes, e.g., research and diagnostic including for purification or immunoprecipitation of KIT from cells, detection of KIT in vitro or in vivo. For isolation and purification of KIT, the kit may contain an anti-KIT antibody coupled to beads (e.g., sepharose beads). Kits may be provided which contain the antibodies for detection and quantitation of KIT in vitro, e.g., in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-ΚΓΓ antibody of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

The present invention also encompasses a finished packaged and labeled

pharmaceutical product. This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial, pre-filled syringe or other container that is hermetically sealed. In one embodiment, the unit dosage form is provided as a sterile particulate free solution comprising an anti-ΚΓΓ antibody that is suitable for parenteral administration. In another embodiment, the unit dosage form is provided as a sterile lyophilized powder comprising an anti- KIT antibody that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus, the invention encompasses sterile solutions suitable for each delivery route. The invention further encompasses sterile lyophilized powders that are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment. Further, the products of the invention include instructions for use or other informational material that advise the physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question, as well as how and how frequently to administer the pharmaceutical. In other words, the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures, and other monitoring information.

Specifically, the invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within said packaging material, wherein said pharmaceutical agent comprises a liquid formulation containing an antibody. The packaging material includes instruction means which indicate how that said antibody can be used to prevent, treat and/or manage one or more symptoms associated with a disease or disorder.

The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including literature references, issued patents, and published patent applications, as cited throughout this application are hereby expressly incorporated herein by reference. It should further be understood that the contents of all the figures and tables attached hereto, as well as the entire contents of U.S. Patent Publication No. 2011/0311538, are expressly incorporated herein by reference.

EXAMPLES

Experimental Protocols for Examples 1-9

Protein Expression and Purification

KITD4-5 Fragment

Soluble KITD4-5 fragment (amino acids 308-514), containing an N-terminal His-tag and TEV cleavage site was expressed in baculovirus Sf9 cells and purified by Ni-affinity followed by size exclusion chromatography (SEC) (Superdex200, GE Healthcare). After partial deglycosilation with endoglycosidase Fl, the KITD4-5 fragment was purified further by anion exchange chromatography (MonoQ, GE Healthcare).

Fabl9, FabUI and Fab79D

Fabl9, Fabl2I and Fab79D Fabs were expressed in Escherichia coli and purified by protein A affinity followed by cation exchange chromatography (Source S, GE Healthcare). Complexes between KITD4-5 fragment and Fab were obtained by mixing purified components in a 1: 1 molar ratio and further purification using SEC (Superdex75, GE Healthcare).

KIT79D Full Length IgG

For construction of full length antibodies, DNA fragments from variable domains of heavy and light chain of KIT79D were subcloned into the antibody cassette vectors, phlgGl (human IgGl backbone) and phlgK (human kappa backbone), that contain human constant regions, respectively. Mouse heavy chain signal peptides were added at the N-terminus of each construct for secretion. Resulting plasmids encoding heavy and light chains of IgG 79D- H (C stands for murine-human chimera, and H stands for full human antibody, respectively) were transfected into 293F cells (Invitrogen). After 3 days, the culture media were harvested, applied to Protein A Sepharose and eluted with 0.2 M Glycine-HCl (pH2.7) followed by neutralization by 1M Tris-HCl (pH8.0). Crystallization and Data Collection

Fab 19 - KITD4-5 and Fab79D - KITD4-5 complexes were crystallized by hanging drop vapor diffusion methods at 21 C. Single crystals for both complexes were obtained by macroseeding. For crystallization of Fabl9-KITD4-5, crystallization buffer containing 13% PEG 3350, 0.5 M MgCk and 0.1 M TrisHCl pH 9.0 was mixed with equal volume (0.6 μΐ) of protein solution (7 mg/ml). Single crystals were dehydrated by transferring into

cryoprotectant solution containing 22% PEG 3350, 0.5 M MgCk, 0.1 M TrisHCl pH 9.0 and 30% ethylene glycol and were incubated over the reservoir of this buffer for 2-3 days.

Crystals were flash frozen in cryoprotectant solution. Crystals of Fab79D-KITD4-5 were obtained by mixing crystallization buffer containing 20-24% PEG 400 and 0.1 M TrisHCl pH 8.2 with protein sample (6.5 mg/ml). Crystals were flash frozen in the reservoir solution supplemented with PEG 400 up to 35%. X-ray diffraction data were collected at the X25 beamline of NSLS, Brookhaven National Laboratory. Data collection statistics are summarized in Table B, infra.

Structure Determination

The structures of Fabl9-KITD4-5 and Fab79D-KITD4-5 complexes were solved by molecular replacement (MR) using PHASER program (McCoy et al. (2007) J App Crystallog 40: 658-674) under the CCP4 software suite (Winn et al. (2011) Acta Crystallogr D 67: 235- 242). To solve the Fabl9-KITD4-5 complex structure, the initial Fab model (from PDB 1FVE, (Eigenbrot et al. (1993) J Mol Biol 229: 969-995) was subdivided into variable (VH/VL) and constant (CHI/CL) fragments and KITm-5 domains (from PDB 2EC8) were divided into individual Ig-like domains which were used as separate search models. The same strategy was used to solve the Fab79D-KITD4-5 complexes structure and Fabl9-KITD4-5 complex as initial model. Coordinates were manually rebuilt and refined using Refmac (Murshudov et al. (1997) Acta Crystallogr D 53: 240-255) and Phenix (Adams et al. (2010) Acta Crystallogr D 66: 213-221). Refinement statistics are summarized in Table B, infra.

Surface Plasmon Resonance (SPR) Analysis

Surface plasmon resonance experiments were performed using a BiaCore T100 instrument at 25°C in the HSP-P+ buffer (GE Healthcare, cat.# BR- 1006-71). KITD4-5 fragment (2.5 μg/ml) was immobilized on a CM5 BiaCore sensor chip (GE) using standard amine coupling.

Optimum coupling was achieved in 10 mM sodium acetate buffer pH 4.5. Surfaces with 3 different concentration of immobilized KITm-5 fragment were obtained by varying contact time (from 50 to 120 s). Non-cross-linked ligand was removed, and unreacted sites were blocked with 1 M ethanolamine, pH 8.5. Purified Fab analytes at series of three fold dilution concentrations (0.12 - 10 nM) were flowed simultaneously over three different

concentrations of immobilized KITm-s and reference surfaces. In order to obtain a reliable signal to noise ratio a longer dissociation time was used for Fabl2I and Fab79D (Figure 12). Data were analyzed using the Biacore T100 Evaluation software.

Phage Display Selection and Characterization

Phage pools consisting of a phage-displayed synthetic antibody library (library F) were cycled through five rounds of selections using KITD4-5 immobilized on 96-well

Maxisorp immunoplates (Thermo Scientific) as antigen, as described previously (Persson et al. (2013) J. Molec Biol 425: 803-811). Culture supernatants of 96 clones from each of rounds 4 and 5 grown in 96-well format were used directly in phage ELISAs to identify clones binding KIT specifically (using BSA as a negative control). KIT-specific clones were subjected to DNA sequence analysis. Unique clones were subjected to competitive ELISAs that allow for affinity estimation and rank ordering amongst clones.

Fab Affinity Maturation

KIT affinity maturation libraries were constructed as described previously (Nelson et al. (2012) Meth Molec Biol 899: 27-41). Briefly, affinity maturation libraries targeting CDR- Hl were constructed by introducing TAA stop codons into CDR-Hl of phagemid Fab 19. The resulting phagemid was used as a template for a mutagenesis reaction that replaced stop codons of CDR-Hl with oligonucleotides mixed in a 70%: 10%: 10%: 10% ratio with targeted the parental nucleotide represented 70%. In this manner, targeted residues were biased towards parental but still allowed all 19 possible substitutions.

Cell Culture, Immunoprecipitation, and Immunoblotting Experiments

NIH-3T3 cells, stably expressing WT-KIT were previously described (Yuzawa et al. (2007) Cell 130: 323-334). Cells were cultured in DMEM (Invitrogen) containing 10% bovine serum and were starved overnight in serum free medium prior to incubation with Fabs and/or SCF stimulation. Cells were incubated with the indicated (Figure 6A) concentrations of Fabs for 5 hours followed by stimulation with SCF. Cells were lysed as previously described (Lax et al. (2002) Molec Cell 10: 709-719) and subjected to immunoprecipitation with anti- KIT antibodies (Yuzawa et al. (2007) Cell 130: 323-334) followed by

immunoblotting with anti-KIT or anti-pY (4G10, Upstate Biotechnology) antibodies.

Ba/F3 Proliferation Assay

Ba/F3 cells were grown in RPMI 1640 medium supplemented with 10% of fetal bovine serum, and 10 ng/mL recombinant murine IL-3. WT and A502,Y503 duplication KIT mutant were cloned into pMSCVpuro vector and transfected into Ba/F3 cells using electroporation. Stable cell lines expressing WT or mutant KIT were selected in the presence of puromycin and IL-3. After establishing stable cell lines, IL-3 was withdrawn and cells expressing WT KIT were supplemented with 250 nM SCF. Ba/F3 cells expressing WT or mutant ΚΓΓ were plated in 6- well plates at 400K/well in 2ml media at day 0. Fab or IgG were added to each well at specified concentrations. 72 hours later, cell number was determined using an Hand Held Automated Cell Counter Sceptor™. Cell number increase is expressed as fold change compared to day 0.

Expression and Purification ofKITp4-5.

Soluble ΚΓΓϋ4-5 fragments were expressed in Sf9 insect cells according to the Bac-to- Bac instruction manual (Invitrogen), using Grace's insect medium supplemented with 10% heat inactivated fetal bovine serum. 72 hours after infection cells were harvested by centrifugation at 500 g and lysed by sonication on ice in lysis buffer (100 mM potassium phosphate buffer pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol and 1% NDP-40). Crude lysate was cleared by centrifugation at 40,000 rpm and filtered through 0.65 μιη PVDF filter, and was applied to Ni-NTA agarose (Qiagen). Ni-NTA beads were washed with 50 column volume (CV) of wash buffer (100 mM potassium phosphate buffer pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol) and eluted with elution buffer (100 mM potassium phosphate buffer pH 8.0, 300 mM NaCl, 250 mM imidazole, 10% glycerol). Eluted proteins were injected onto a Hiload 26/600 Superdex 200 (GE) equilibrated with 10 mM Tris pH 8.0, 200 mM NaCl. Fractions corresponding to KITD4-5 fragment were combined and concentrated using Vivaspin concentrator (GE) with MWCO 10 kDa. The concentrated sample was deglycosylated with recombinant endo-glycosylase Fl at a final 1:50 w/w ratio, for 12 hours at room temperature. Simultaneously the 6His tag was cleaved by adding 1: 100 w/w ratio of recombinant TEV protease that preceded deglycosylation and TEV treatment of KITD4-5 that was purified by anion exchange chromatography at 16/10 MonoQ column using 40 CV gradient from Buffer A (10 mM BIS-Tris pH 6.5) to 50% of Buffer B (10 mM BIS-Tris pH 6.5, 1 M NaCl). Fractions containing KITm-s fragment were combined and concentrated. For SPR analysis the buffer of the KITD4-5 was additionally exchanged to an HSP-P+ buffer using a Superdex75 10/300 GL (GE).

Fab Expression and Purification

Synthetic codon optimized bicistronic DNA of KTN37 Fab was cloned into pET26 vector using Ncol and Sall/Xhol restriction sites. A His-tag was introduced into the C- terminus of heavy chain of KTN37 Fab. Phagemids as described in Nelson et al. (Meth Molec Biol (2012) 899: 27-41) were used for Fabl9, Fabl2I and Fab79D expression.

BL21(DE3) was transformed with appropriate phagemid or pET26-KTN37 and single colony was used for expression in Terrific Broth (TB) media using the autoinduction protocol (Studier et al. (2005) Protein Expres Purif 41: 207-234). After overnight expression at 22°C cells were harvested and flesh frozen in appropriate lysis buffer containing IxTBS (Fabl9, Fabl2I and Fab79D) or 100 mM potassium phosphate buffer pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol (KTN37 Fab). Cells were lysed using a French press and crude lysate was cleared by centrifugation at 20,000 rpm and filtration through 0.65 μιη PVDF filter. Cleared lysates of Fabl9, Fabl2I or Fab79D were applied to Protein A sepharose (Invitrogen) equilibrated with IxTBS buffer. Unbound proteins were washed out with 50 CV of IxTBS buffer. Fabs were eluted with 100 mM glycine-HCl buffer pH 3.2 and immediately neutralized with 1 M Tris-HCl pH 8.0 buffer. Cleared lysate of KTN37 Fab was applied to Ni-NTA agarose (Qiagen). Unbound proteins were washed out with 50 CV of wash buffer (100 mM potassium phosphate buffer pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol). KTN37-Fab was eluted with elution buffer (100 mM potassium phosphate buffer pH 8.0, 300 mM NaCl, 250 mM imidazole, 10% glycerol) and applied onto a Hiload 26/600 Superdex 200 equilibrated with IxTBS. The buffers of all Fabs solutions were exchanged to a 10 mM sodium acetate pH 5.0 buffer using HiPrep 26/10 Desalting column, and applied to Source S HR 16/10 column equilibrated with the same buffer. Fabs were eluted with 20 CV gradient from 10 mM sodium acetate pH 5.0 buffer to 10 mM sodium acetate pH 5.0, 1 M NaCl. Fractions containing assembled Fabs were neutralized with 1 M Tris-HCl pH 8.0 and concentrated. For the SPR analysis, the buffers of KITD4-5 was additionly exchanged to an HSP-P+ buffer using a Superdex 75 10/300 GL (GE Healthcare). Structure Determination and Refinement

For the Fabl9-KITD4-5 complex a native dataset at X25 beamline NSLS was collected.

This crystal diffracted to 2.4 A resolution (Table B, infra), with unit cell parameters of a = 169.6 A, b = 49.0 A, c = 107.0 A, cc=^=90°, β=122.4° in C2 space group. For the Fab79D- KITD4-5 complex a native dataset at beamline X29A at NSLS was collected. The Fab79D-

KITD4-5 complex crystal diffracted to 2.4 A resolution (Table B, infra) and had unit cell parameters of a=b= 95.9 A, c= 322.6 A,

β=122.4° in space group P6s22. The processed datasets for both structures were strongly anisotropic (see Figure 16 and Figure 17) and were corrected for anisotropy using the Diffraction Anisotropy Server at UCLA

(http://services.mbi.ucla.edu/anisoscale/) (Strong et al. (2006) 103: 8060-8065).

Fabl9-KITD4-5 Complex

Matthews coefficient analysis suggested the highest probability of 1 Fabl9-KITD4-5 complex molecule per asymmetric unit. Molecular replacement was conducted using Phaser (McCoy et al. (2007) J Appl Crystallog 40: 658-674) and following ensembles used as search models: ensemble 1 - Fv region of humanized anti-pl85HER2 antibody 4D5 (PDB 1FVE: chain A, residues 1-104 and chain B, residues 1-122); ensemble 2 - KIT D4 (PDB 2EC8, residues 312-408); ensemble 3 - constant domain of the heavy chain of humanized anti- pl85HER2 antibody 4D5 (PDB 1FVE, chain B, residues 121- 223); ensemble 4 - constant domain of the light chain of humanized anti-pl85HER2 antibody 4D5 (PDB 1FVE, chain A, residues 110-214); and ensemble 5 - KIT D5 (PDB 2EC8, residues 410-504) (Eigenbrot et al. (1993) J Molec Biol 229: 969-995; Yuzawa et al. (2007) Cell 130: 323-334). A single solution was found using this strategy. Standard refinement and model building techniques using Refmac (Murshudov et al. (1997) Acta Crystallogr D 53: 240-255) and Coot (Emsley et al. (2010) Acta Crystallogr D 66: 486-501) were then followed. Phenix refine was used for the last stage of refinement (Afonine et al. (2012) Acta Crystallog D 68: 352-367).

Fab79D-KITD4-s Complex

The structure of Fab79D-KITD4-5 complex was solved by molecular replacement using Phaser (McCoy et al. (2007) J Appl Crystallogr 40: 658-674). A clear molecular replacement solution was found using the refined Fabl9-KITD4-5 complex structure as a search model. The Fabl9-KITD4-5 complex structure (from this work) was split into four search models:

ensemble 1 - variable fragment of Fab 19 (chain A residues 3-108 and chain B residues 1- 120) and KIT D4 fragment (chain C residues 310-409); ensemble 2 - Ch fragment (chain B residues 121-217); ensemble 3 - CI fragment (chain A residues 113-209); and ensemble 4 - KIT D4 fragment (chain C residues 410-504). Strong electron density has been seen at the C- terminus of the light chain of Fab79D and was assigned to the Flag-tag, but because electron density is missing in the linker region between Flag-tag and light chain, precise determination of the registry for the Flag-tag is impossible. The same refinement strategy as was used for Fab 19- KITD4-5 complex structure, was used for refinement of Fab79D-KITD4-5 complex structure.

Data collection and refinement statistics for both structures are summarized in Table B, infra, and example electron densities are presented in Figure 14 (Fab 19 complex) and Figure 15 (Fab79D complex).

Competitive ELISA

Briefly, supernatant from clones grown in 96-well format was incubated for 1 h with specifiedconcentrations of soluble antigen before being applied to KITD4-5 immobilized to Maxisorpimmunoplates for 15 min. Signal inhibition was compared to phage incubated in the absence of solubleantigen. Multi-point ELISAs involving normalization of purified phage particles (grown in 30mlcultures and quantified using A268) and 8, 4-fold serial dilutions used for ELISAs were also used to rank variants.

Fab Affinity Maturation

Affinity maturation libraries targeting CDR-Ll were constructed by introducing TAA stopcodons into CDR-Ll and CDR-Hl of phagemid Fabl9. The resulting phagemid was used for 3independent mutagenesis reactions to replace stop codons of CDR-Hl with Fab 19 CDR- Hl containing either the double S31VS33M substitution, the S3 IV single substitution, or the S33M single substitution. CDR-Ll was targeted simultaneously in all 3 libraries by replacing CDR-Ll stop codons with 4-7 NNK codons (representing all 20 amino acids). The 3 libraries were mixed together before selections.

Example 1: Isolation, Characterization and Crystal Structure Determination of Fabs

The two membrane proximal Ig-like domains of KIT (KITD4-5 fragment, see Figure 1A) determined structurally to be critical for KIT activation (Yuzawa et al. (2007) Cell 130: 323-334) were used as an antigen to isolate binding Fabs from a naive phage-displayed library (library F) containing more than 10¹⁰unique clones (Nelson et al. (2012) Meth Molec Biol 899: 27-41; Persson et al.( 2013) J Molec Biol 425: 803-811). The binding properties of the phage-derived Fabs were compared to the binding properties of a murine monoclonal anti-KIT antibody designated KTN37 that was obtained by immunization of mice with the same antigen. A variety of in vitro binding experiments using 3T3 cells ectopically expressing WT KIT demonstrated that the phage-derived Fabs and the KTN37 mAb bind specifically to D4 of recombinant isolated ΚΓΓ or to native KIT molecules expressed on the cell surface of live cells. The structure of one of the most potent phage-derived Fab, designated Fab 19 (Figure 8) in complex with ΚΓΓϋ4-5 was further analyzed by X-ray crystallography.

The Structure of Fabl9-KITp4-5 Complex

A purified complex composed of Fab 19 together with Krfm-s was subjected to extensive screening for crystal growth and further optimization. Crystals that belong to the C2 space group with a single 1: 1 complex of KITD4-5 and Fab 19 in the asymmetric unit were obtained. The structure of this complex was determined to 2.4 A resolution (see experimental procedures and Table B, infra and Figure IB for details). The overall structure of KITD4-5 - bound to Fab 19, is very similar to the structures of these two Ig-like domains observed previously as part of the structures of full length extracellular region of KIT alone, or in complex with SCF (PDB 2EC8 and PDB 2E9W (Yuzawa et al. (2007) Cell 130: 323-334)). Superposition of individual D4 and D5 from Fabl9-KITD4-5 complex structure with corresponding domains of KIT ectodomain structure (PDB ID 2EC8) revealed root mean square deviation (r.m.s.d.) values of 0.65 A for 96 and 59 Ccc residues in D4 and D5 respectively. The structure revealed Fab 19 binding exclusively to D4 of KIT with a buried surface of 1029 A2 on the D4 side of the interface (Figure IB and Table A).

The interface between Fabl9 and KIT_D4_₅, was calculated using PDBsum web service (Laskowski (2007) Bioinformatics 23: 1824-1827) and is summarized in Table A. The number of residues involved in interface formation is designated 'Nres. The number of residues involved in hydrogen bonding formation is designated N_HB- Table A

Nearly the entire β sheet of D4 (one out of two β sheets in Ig like domain), including βΑ, βΒ, βΕ, βϋ as well as the AA', A'B, EF and DE loops was buried under the Fabl9 surface (Figures 1C and 9). The overall shape complementarity (SC) parameter for the

Fabl9-KITD₄-5 interface was 0.70, slightly higher than observed typically (0.65 to 0.68) for an antibody-antigen binding interface (Lawrence et al. (1993) J Molec Biol 234: 946-950). A higher SC parameter correlates to higher affinity or more convex shape of the antibody- antigen interface; indeed, similar or even higher SC values (in the range from 0.7 to 0.75) have been observed for therapeutic antibody-antigen complexes such as cetuximab (Li et al. ( 2005) Cancer Cell 7: 301-311), trastuzumab (Cho et al. (2003) Nature 421: 756-760) and pertuzumab (Franklin et al. (2004) Cancer Cell 5: 317-328).

The majority of the contacts were made by the heavy chain of the Fab (800 A ° 2 versus

283 A ° 2 for the light chain, see Figure 1C, 9 and Table A, supra) with most key interactions mediated by the complementary-determining region (CDR) loops of the Fab including all 3 CDRs of the heavy chain and L2 and L3 of the light chain. The majority of the specificity- determining contacts came from CDRs H2 and H3 (Figure 2). The AA' loop of D4 (residues Pro317^D4 - Asn320^D4, where ^D4 stands for the D4 domain of KIT) was buried in the cavity created by the heavy chain CDRs HI and H2 (Figure 2A and B). In particular, CDR H2 created a pronounced hydrophobic pocket (Tyr52 , Tyr54 , Ser55 and Tyr57 with standing for heavy chain) that interacted with Pro317^D4, Met318^m and Asn320^D4 of the AA' loop of the D4 (Figure 2B and 9). Ser30^H and Ser31^H of the CDR HI made van der Waals contacts with Ile319^D4 and glycosylated Asn320^D4; Ser30^H was also located within hydrogen bond distance from the main chain of Asn320^D4 (Figure 2A and S2). Tyr32^Hfrom CDR HI also interacted with the A'B loop of D4 (Asn330^D4 and Glu329^D4) through van der Waals interactions and hydrogen bonding with the main chain of Asn330^D4. CDR H2 makes additional interactions with βΒ (Tyr52^H and Tyr57 interact with Ile334 and Glu336 , respectively), βΕ (Tyr57 makes a hydrogen bond with Arg372^m) and DE loop (Tyr59^H makes van der Waals contacts with Lys364^D4). CDR H3 makes extensive polar and hydrophobic contacts with βΒ, βΕ, βϋ and A'B and EF loops of D4 (Figures 2C and 9). Moreover, TyrlOl of CDR H3 made a π-π staking with His378^D4 while also interacting with Thr380^D4, Lys358^D4 and Asp332^m Hisl02^H and

Vall00^H of CDR H3 were additional key residues, making contact with Glu360^D4, Glu376^D4, His378^D4 and Val331^m, Asp332^m Arg98^H, located at the base of CDR H3, formed a salt bridge with Glu329^m. Finally, CDRs L2 (Tyr49^L and Leu54^L) and L3 (Trp91^L, Val93^L and His94^L) of the light chain were involved in interactions with βϋ (Tyr362^D4), DE (Glu366^D4) and EF (Arg381^m) loops of the D4 (Figures 2D, 3A and 9).

Example 2: Fabl9 Contacts Critical for KIT Receptor Inhibition

D4-D4 homotypic interactions mediated by two salt bridges between Arg38lD4 and G1U386D4, both located in the EF loop (Figure 3B), have been shown to be critical for proper ligand dependent receptor activation (Yuzawa et al. (2007) Cell 130: 323-334). Analysis of the Fabl9-KITD4-5 complex structure revealed Arg381^D4 makes contact with CDR L2 of Fabl9 (Figure 3A); the Arg381^D4 side chain makes hydrogen bonds with the side chain of Tyr49 and the main chain of Leu54 . In addition, TyrlOl of CDR H3 makes contact with Thr380^D4 (Figure 2C) of the EF loop, which is involved in mediating D4-D4 homotypic interactions. Figure 3C shows the overlap (in yellow) on the D4 surface between Fab 19 interface and interface of D4-D4 homotypic interactions. The structure revealed that contacts between Fabl9 and D4 of KIT occluded Axg \^m from forming a salt bridge with Glu 386 of D4 from a neighboring KIT receptor, thereby preventing proper lateral association between membrane proximal domains. As a consequence, interactions between the cytoplasmic domains, transphosphorylation and KIT activation were inhibited (see below).

Example 3: Affinity Maturation of Fab 19

In order to improve the binding affinity of Fab 19 to D4, affinity maturation was performed in a twostep process. The first step was performed prior to solving of the Fab 19- KITD4-5 complex structure and involved soft randomization of individual CDRs wherein targeted residues were likely to be kept parental thereby minimizing the likelihood of changing epitopes during Fab maturation. The 4 CDR regions into which diversity was introduced in library F, CDR L3 and all heavy chain CDRs, were targeting individually in affinity maturation library design and resulted in the isolation of numerous variants from all 4 libraries (data not shown). Variants from the CDR HI library showed strong improvement over the parental Fab 19 and in particular, Fab 121, estimated to have the strongest binding affinity, was chosen for further affinity maturation with the aid of the newly solved Fab 19- KITD4-5 complex (Figure lOA-C).

The structure of the Fabl9-KITD4-5 complex was used to guide affinity maturation library design after analysis of the antibody-antigen interface revealed that while the heavy chain of Fab 19 makes extensive contacts with D4, light chain interactions were few and weak (Figure ID). In particular, no contacts were observed between CDR LI and D4 (Figure 5B).

Considering that the light chain makes most of the contact with Arg381^D4, a residue that is important for formation of D4 homotypic contacts for receptor activation, affinity maturation libraries targeting CDR LI were designed. Length variation between 4-7 residues was incorporated into library design in addition to allowing for possible CDR HI S3 IV and/or S33M substitutions found in the parental Fabl2I CDR HI template. Eight unique Fabs (Fab79A-H) were isolated with CDR LI lengths ranging from 4-7 residues, suggesting that increased length allowed additional contacts with D4 (Figure 10D). Interestingly, all clones were found to contain double S3 IV S33M substitutions indicating the importance of these nucleophilic to hydrophobic substitutions.

Example 4: Binding of Anti-D4 Fabs to KITD4-5 Fragment

SPR analysis was used to quantitatively characterize the kinetics and dissociation constants of different generations of affinity matured anti-D4 Fabs (Fabl9, Fabl2I and Fab79D). As a control, the Fab of the murine antibody KTN37 was used (see below). Purified KITD4-5 fragment, was covalently attached onto a CM5 biosensor chip and serial dilutions of each Fab were flowed over the biosensor surface to reveal the binding kinetics (Figure 4). Values for the association and dissociation rates as well as Gibbs free energy showed that the initial phage-derived synthetic Fabl9 possessed high binding affinity with a KD value of 0.63 nM, similar to the affinity of the mouse-derived KTN37 Fab, which had a KD value of 0.25 nM (Figure 4B). Although Fab 19 and KTN37 Fab had similar affinities, KTN37 bound to KIT with a high association rate but relatively fast dissociation rate, while Fab 19 showed slower association and dissociation rates.

In two steps of affinity maturation, improved binding affinity by two orders of magnitude were observed, bringing binding affinity from the subnanomolar (KD= 0.63 nM for Fabl9) to the picomolar range (KD= 6.4 pM for Fab79D). Substitution of only two residues, Ser31 and Ser33 , to Val and Met respectively, was observed after the first affinity maturation step. The S3 IV substitution allowed improved association through increased hydrophobicity and improved van der Waals interactions (See below and Figure 11 for details). Surprisingly, residue 33 did not make contact (see below) with D4 in either structure (Fabl9-KITD4-5 or Fab79D-KITD4-s), however, substitution of S33M likely stabilized the CDR HI conformation orientation towards D4 (see Figure 11). It is worth noting that each step of affinity maturation improved binding energy by 1 or 2 kcal/mol, corresponding to weak interactions such as hydrogen bonding, but had a strong impact on their binding affinity. The difference in Gibbs free energy of complex formation correlates well with the structural analyses that showed small but significant differences in critical regions of the two complexes (see below).

Example 5: Structure of Fab79D-KITD4-5 Complex

In order to understand how affinity maturation improved the binding properties of Fab79D compared with Fab 19 and to confirm that the binding epitope remained unchanged, crystallization of the Fab79D-KITD4-5 complex was performed. The structure was solved by molecular replacement using the Fab 19-11 KITm-5 complex as a search model. Data collection and refinement statistics are presented in Table B.

Comparison between individual domains of the two structures of Fab 19 and Fab79D in complex with KITm-5 fragments showed them to be highly similar with the biggest difference in the elbow angle between D4 and D5 and the elbow angle between the variable and constant domains of the Fabs. Superposition of the constant domains of Fab 19 and

Fab79D and KIT D5 domains within complex structures revealed r.m, s.d of 0.41 A and 0.74

A for Ccc residues respectively. Similarly, variable domains and D4 were not altered significantly with r.m.s.d values of 0.55 A for 279 Ccc residues of the VL, VH domains and D4 (Figure 5A). As expected the biggest difference between the two complexes was found in the CDR LI loop, which was targeted with length diversity during affinity maturation. Figure 5A shows that the LI loop of Fab79D moved towards the D4 domain within the Fab79D- KITD4-5 complex structure and unlike Fab 19, made contact with βϋ of D4 (Figure 5C and 13); Arg31^L and Asn32^L of Fab79D were located within hydrogen bonding distance of the main chain of Prc^S⁰⁴ and side chain of Glu360^D4 respectively. This CDR LI loop extension, so evident upon complex structure comparison, appears to be responsible for the increased binding affinity of Fab79D towards KIT D4. Example 6: KIT Autophosphorylation is Inhibited by Anti-D4 Antibodies in Living Cells

The crystal structure of Fabl9-KITD4-5 and Fab79D-KITD4-5 complexes revealed significant overlap between the Fab binding epitope and the D4-D4 homotypic interface (Figure 3C), important for KIT activation, suggesting that binding of these Fabs likely interferes with KIT receptor autophosphorylation. To test for KIT inhibition, NIH-3T3 cells expressing wt KIT receptor were incubated for 5 hours with varying concentrations of Fabs or IgGs prior to SCF stimulation. All three generations of synthetic Fabs together with Fab KTN37 were tested along with the IgG version of Fab79D (IgG 79D) and IgG KTN37. As predicted from the structure, SCF- stimulated autophosphorylation of KIT was strongly inhibited upon binding of anti-D4 Fabs and IgGs (Figure 6A).

During the two steps of affinity maturation, the inhibitory properties of the anti-D4 Fabs were improved significantly; KIT inhibition by Fab 19, the parental synthetic Fab, occurred at 50 nM whereas the last generation Fab, Fab79D, inhibited KIT at 5 nM

suggesting that increased affinity correlated to increased KIT inhibition (Figure 6A and 4B). Consistent with this, Fabl2I appears to be more effective at KIT inhibition than Fabl9 but weaker than Fab79D. The bivalent IgG format confers avidity effects to a Fab that are evident upon testing IgG KTN37, whose effectiveness at blocking KIT autophosphorylation could be seen even at 0.5 nM compared to the 50 nM level required for Fab KTN37 (Figure 6A). A gain in KIT inhibition from avidity could also be seen upon conversion of Fab79D to IgG 79D (5 nM for Fab79D and 1 nM for IgG 79D).

Example 7: Anti-D4 Antibodies Efficiently Inhibit Proliferation of KIT-Dependent Ba/F3 Cells

The effect of the Fabs and IgGs on KIT mediated cell proliferation was examined. Stable Ba/F3 cell lines were constructed expressing wt KIT (Ba/F3 KIT^wt) or an oncogenic AY502,503 duplication KIT mutant (Ba/F3 _KIT^AY502'^503dup). The parental Ba/F3 cells are an interleukin-3 (IL-3) dependent murine pro-B-cells lacking endogenous KIT expression. Upon exogenous WT KIT expression, Ba/F3 cells become dependent on SCF stimulation of these cells. Moreover, expression of constitutively active KIT (such as the AY502,503 duplication in D5) induces transformation of these cells (Guo et al. (2007) Cancer Res 13: 4874-4881). Incubation of these lines with anti-D4 Fabs and IgGs revealed varying cell proliferation inhibition of the D4 binders that correlated with results seen in the KIT autophosphorylation inhibition experiments (Figure 6 and Figure 7). Like in the inhibition of KIT

autophosphorylation experiment, Fab 19 could inhibit Ba/F3 KIT wt cell proliferation only at the highest concentration (500 nM) whereas subsequent generations of anti-D4 Fab showed significant improvements to both phosphorylation and proliferation inhibition; indeed, Fab79D showed significantly improved inhibitory properties that could inhibit cell growth at 50 nM. KTN37 Fab showed an inhibitory effect at 200 nM, and avidity effects increased upon testing of IgG KTN37, whereupon cell proliferation inhibition could be seen at 1 nM.

As with the Ba/F3 KITwt cells overexpressing wt KIT, significant improvements to cell proliferation inhibition of unstimulated Ba/F3 jQT^AY502~3dup could also be seen with successive generations of affinity matured Fabs (Figure 7). As observed with Ba/F3 KIT^wt cells, Fab79D inhibited cell proliferation at 5 nM, Fab KTN37 inhibited cell proliferation at 50 nM, IgG KTN37 inhibited cell proliferation at 1 nM, IgG 79D inhibited cell proliferation at 10 nM, and Fab79D inhibited cell proliferation at 5 nM (Figure 7).

Example 8: Impact of S31V and S33M Substitutions

Fab 121 was amongst the strongest Fab binders selected during first generation of affinity maturation and contained two substitutions in CDR HI: S31/V and S33/M. The same substitutions in CDR HI were permitted during the second generation of structurally guided affinity maturation targeting CDR LI and resulting in the isolation of Fabs all containing both substitutions (S31/V and S33/M). Analysis using structural alignments of CDR HI from structures of Fab 19 and Fab79D complexes is depicted in Figure 11 with Fab 19 colored with cyan and Fab79D in blue. Substitution of S31/V increased hydrophobicity of this residue and allowed additional van der Waals interactions with Val323D4 explaining the increased affinity to KIT D4. Surprisingly no contacts between Ser33H (for Fab 19) or Met33H

(Fab79D) and KIT D4 were found. Substitution of Ser33 to Met stabilizes conformation of CDR HI by additional hydrophobic contacts between Met33H and Tyr51H (Figure 11B). This conformation of CDR HI would be poised for binding to KIT D4 and wouldn't require additional energy for stabilization upon binding as in the case of Fab 19. In order to understand the role of individual CDR HI mutations, Fabl9-S31V and Fab 19- S33M single mutants were generated to analyze their binding affinities to the KITD4- 5 fragment. Both mutants demonstrated increased binding affinities compared to those of parental Fab 19:

Table B: Data collection and refinement statistics

Model quality

Rmsd bond length (A) 0.004 0.004

Rmsd bond angles (°) 0.770 0.750 β-factor (A)

Average overall 51.9 52.42

KIT D4 41.8 44.96

KIT D5 49.4 63.01

Fab light chain 61.4 59.51

Fab heavy chain 49.4 44.99

N-acetylglucosamine 31.7 61.8

Water 39.6 39.59

Fab elbow angle (°) 143 163

MolProbity

Ramachandran plot ( ) 95.95/0.34 95.18/0.16

favored/outliers

MolProbity score 2.29 (82^th percentile) 2.49 (87^th percentile)

PDB Accession Code 4K94 4K9E

Values in parentheses indicate the highest resolution bin

Conclusion to Examples 1-8

Tyrosine kinase inhibitors were applied successfully in the clinic for treating cancer patients whose tumors were driven by activated RTKs. Indeed, both Gleevec and Sutent were applied successfully for treatment of GIST driven by activated KIT. While most ΚΓΓ driven GIST patients respond well when treated with tyrosine kinase inhibitors, sometimes with several years of remission, eventually most cancers relapse because of drug resistance. An alternative and complementary approach is to develop therapeutic monoclonal antibodies that target the extracellular region of KIT.

Rational design of a drug that target the extracellular region of KIT became possible upon solving of the crystal structure and elucidating the mechanism of ligand induced or oncogenic KIT activation. Crystal structures of the extracellular regions of KIT before and after ligand stimulation (Yuzawa et al. (2007) Cell 130: 323-334) strongly emphasized the role of membrane proximal domain homotypic interactions in receptor activation. Upon ligand binding, the weak homotypic interactions between membrane proximal D4 and D5 allow precise positioning of the two C-terminal regions of the receptor ectodomains in a manner and distance important for positioning of the TM domains in the correct orientation that enable activation of the cytoplasmic tyrosine kinase domain (Yang et al. (2008) Proc Natl Acad Sci USA 105: 7681-7686; Yang et al. (2010) Proc Natl Acad Sci USA 107: 1906- 1911; Arkhipov et al. (2013) Cell 152: 557-569;

Endres et al. (2013) Cell 152: 543-556). Moreover, disruption of homotypic contacts in the membrane proximal domains of KIT (Yuzawa et al. (2007) Cell 130: 323-334), PDGFR (Yang et al. (2008) Proc Natl Acad Sci USA 105: 7681-7686) and VEGFR2 (Yang et al. (2010) Proc Natl Acad Sci USA 107: 1906-1911) strongly impairs receptor activation.

Described here is the isolation and maturation of an antibody directed against KIT D4 that strongly impairs receptor activation and cell proliferation to a level that can be used in cancer therapy. Phage display was combined with structural guidance to develop anti-KIT antibodies. Structural analysis of the Fabl9-KITD4-5 complex allowed suboptimal contacts to be determined, thereby permitting facile and focused affinity maturation library design. Indeed, structural analysis revealed that most contacts were mediated by the heavy chain of Fab 19, whereas contacts between the light chain and D4 of KIT were crucial for inhibition of KIT receptor. Considering the importance of the light chain contribution to KIT inhibition, affinity maturation libraries were directed towards CDRL1, lacking any contact between parental Fab 19 and KIT D4, that included length variation in attempt to create contacts. This structure guided strategy proved successful as significant improvement to inhibitory properties of the final variant (Fab79D) could be seen, especially when compared to those of the affinity matured variant isolated in the absence of structural information (Fabl2I).

In addition to design of affinity maturation libraries, structure can provide valuable insight into the precise nature of inhibition. The crystal structures of these inhibitory Fabs in complex with ΚΓΓϋ4-5 revealed clear steric blocking of homotypic contacts between the membrane proximal domains of KIT by the Fabs that led to inhibition of receptor autophosphorylation and activation.

In summary, the antibodies of the invention affect proliferation at nanomolar concentrations and can therefore be used for anticancer therapy and to help overcome resistance that frequently occurs in patients treated with tyrosine kinase inhibitors.

Furthermore, these antibodies can be used in combination with tyrosine kinase inhibitors or as antibody:toxin conjugates to allow usage of lower doses to treat disease and/or delay or prevent the occurrence of resistance to the therapeutic.

Exemplary antibodies of the invention are provided in Tables 1, 2, 3, and 4, below Table 1: List of Sequences for Antibodies, and Antigen-Binding Portions Thereof, of the Invention

Fab 121; KTN0044H

SEQID Light chain CDR2 SASSLYS

NO.8 (Kabat and Chothia):

Fabl9;Fabl9-S31V;

Fabl9-S33M; Fabl9- S31VS33M;Fabl2F;

Fabl2G; Fabl2H;

Fab 121; Fab79A,

Fab79B; Fab79C;

Fab79D; Fab79E;

Fab79F; Fab79G;

Fab79H; KTN0044H;

KTN0117H

SEQID Light chain CDR3 QQWAVHSLIT

NO.9 (Kabat, Chothia and

IMGT): Fabl9; Fabl9- S31V;Fabl9-S33M;

Fabl9-S31VS33M;

Fabl2F; Fabl2G;

Fabl2H; Fab 121;

Fab79A; Fab79B;

Fab79C; Fab79D;

Fab79E; Fab79F;

Fab79G; Fab79H;

KTN0044H;

KTN0117H

SEQID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSA NO.10 region: SSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQG

Fabl9;Fabl9-S31V; TKVEIKR

Fabl9-S33M; Fabl9- S31VS33M;Fabl2F;

Fabl2G; Fabl2H;

Fab 121

SEQID Light chain constant TVAAPSVFIFPPSDSQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS NO.11 region, including Flag- QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN

tag: RGECGGSDYKDDDDK

Fabl9;Fabl9-S31V;

Fabl9-S33M; Fabl9- S31VS33M;Fabl2F;

Fabl2G; Fabl2H;

Fab 121; Fab79A,

Fab79B; Fab79C;

Fab79D; Fab79E;

Fab79F; Fab79G;

Fab79H

SEQID Light chain, including DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSA NO.12 Flag-tag: SSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQG

Fabl9;Fabl9-S31V; TKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDN Fabl9-S33M; Fabl9- ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS S31VS33M;Fabl2F; PVTKSFNRGECGGSDYKDDDDK

Fabl2G; Fabl2H;

Fab 121

SEQID Heavy chain CDR1 VYSMH

NO.13 (Kabat): Fabl9-S31V

SEQID Heavy chain variable EVQLVESGGGLVQPGGSLRLSCAASGFNISVYSMHWVRQAPGKGLEWVASI NO.14 region: YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fabl9-S31V ALDYWGQGTLVTVSS

SEQID Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNISVYSMHWVRQAPGKGLEWVASI NO. 15 Fabl9-S3 IV Heavy YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH chain ALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC

SEQ ID Heavy chain CDR1 SYMMH

NO. 16 (Kabat): Fabl9-S33M;

Fabl2F

SEQ ID Heavy chain variable EVQLVESGGGLVQPGGSLRLSCAASGFNISSYMMHWVRQAPGKGLEWVASI NO. 17 region: YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fabl9-S33M ALDYWGQGTLVTVSS

SEQ ID Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNISSYMMHWVRQAPGKGLEWVASI NO. 18 Fabl9-S33M YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

ALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC

SEQ ID Heavy chain CDR1 VYMMH

NO. 19 (Kabat): Fabl9- S31VS33M; Fabl2G;

Fab 121; Fab79A;

Fab79B; Fab79C;

Fab79D; Fab79E;

Fab79F; Fab79G;

Fab79H; KTN0117H

SEQ ID Heavy chain variable EVQLVESGGGLVQPGGSLRLSCAASGFNISVYMMHWVRQAPGKGLEWVASI NO. 20 region: YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fabl9-S31VS33M; ALDYWGQGTLVTVSS

Fab 121; Fab79A;

Fab79B; Fab79C;

Fab79D; Fab79E;

Fab79F; Fab79G;

Fab79H

SEQ ID Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNISVYMMHWVRQAPGKGLEWVASI NO. 21 Fabl9-S31VS33M; YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fab 121; Fab79A; ALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP Fab79B; Fab79C; VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH Fab79D; Fab79E; KPSNTKVDKKVEPKSC

Fab79F; Fab79G;

Fab79H

SEQ ID Heavy chain variable EVQLVESGGGLVQPGGSLRLSCAASGFNIDSYMMHWVRQAPGKGLEWVASI NO. 22 region: YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fabl2F ALDYWGQGTLVTVSS

SEQ ID Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIDSYMMHWVRQAPGKGLEWVASI NO. 23 Fabl2F YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

SEQ ID Heavy chain CDR1 AYMMH

NO. 24 (Kabat): Fabl2H

SEQ ID Heavy chain variable EVQLVESGGGLVQPGGSLRLSCAASGFNIDVYMMHWVRQAPGKGLEWVASI NO. 25 region: YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fabl2G ALDYWGQGTLVTVSS

SEQ ID Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIDVYMMHWVRQAPGKGLEWVASI NO. 26 Fab 12G YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

SEQ ID Heavy chain variable EVQLVESGGGLVQPGGSLRLSCAASGFNIDAYMMHWVRQAPGKGLEWVASI NO. 27 region: YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

Fabl2H ALDYWGQGTLVTVSS

SEQ ID Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIDAYMMHWVRQAPGKGLEWVASI NO. 28 Fabl2H YPYSGYTYYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARYVYH

SEQ ID Light chain CDR1 RASQIRHRLRRAVA

NO. 29 (Kabat and Chothia):

Fab79A

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQIRHRLRRAVAWYQQKPGKAPKLLI NO. 30 region: YSASSLYSGVPSRFSGSRSGTDFTLTI SSLQPEDFATYYCQQWAVHSLITF

Fab79A GQGTKVEIKR

SEQ ID Light chain, including DIQMTQSPSSLSASVGDRVTITCRASQIRHRLRRAVAWYQQKPGKAPKLLI NO. 31 Flag-tag: YSASSLYSGVPSRFSGSRSGTDFTLTI SSLQPEDFATYYCQQWAVHSLITF

Fab79A GQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain CDR1 RASQIRKVAVA

NO. 32 (Kabat and Chothia):

Fab79B

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQIRKVAVAWYQQKPGKAPKLLIYSA NO. 33 region: SSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQG

Fab79B TKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQIRKVAVAWYQQKPGKAPKLLIYSA NO. 34 Flag-tag: SSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQG

Fab79B TKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain CDR1 RASQRYNTRPMAVA

NO. 35 (Kabat and Chothia):

Fab79C

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQRYNTRPMAVAWYQQKPGKAPKLLI NO. 36 region: YSASSLYSGVPSRFSGSRSGTDFTLTI SSLQPEDFATYYCQQWAVHSLITF

Fab79C GQGTKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQRYNTRPMAVAWYQQKPGKAPKLLI NO. 37 Flag-tag: YSASSLYSGVPSRFSGSRSGTDFTLTI SSLQPEDFATYYCQQWAVHSLITF

Fab79C GQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain CDR1 RASQRGLRNVAVA

NO. 38 (Kabat and Chothia):

Fab79D; KTN0117H

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQRGLRNVAVAWYQQKPGKAPKLLIY NO. 39 region: SASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFG

Fab79D QGTKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQRGLRNVAVAWYQQKPGKAPKLLIY NO. 40 Flag-tag: SASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFG

Fab79D QGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain CDR1 RASQRGRTAVA

NO. 41 (Kabat and Chothia):

Fab79E SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQRGRTAVAWYQQKPGKAPKLLIYSA NO. 42 region: SSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQG

Fab79E TKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQRGRTAVAWYQQKPGKAPKLLIYSA NO. 43 Flag-tag: SSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQG

Fab79E TKVEIKRTVAAPSVFIFPPSDSQLKSGTASWCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQRGARSAVAWYQQKPGKAPKLLIYS NO. 44 region: ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQ

Fab79F GTKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQRGARSAVAWYQQKPGKAPKLLIYS NO. 45 Flag-tag: ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQ

Fab79F GTKVEIKRTVAAPSVFIFPPSDSQLKSGTASWCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain CDR1 RASQPFRRVAVA

NO. 46 (Kabat and Chothia):

Fab79G

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQPFRRVAVAWYQQKPGKAPKLLIYS NO. 47 region: ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQ

Fab79G GTKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQPFRRVAVAWYQQKPGKAPKLLIYS NO. 48 Flag-tag: ASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQWAVHSLITFGQ

Fab79G GTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGECGGSDYKDDDDK

SEQ ID Light chain RASQNGNVRI SAVA

NO. 49 CDRl(Kabat and

Chothia): Fab79H

SEQ ID Light chain variable DIQMTQSPSSLSASVGDRVTITCRASQNGNVRISAVAWYQQKPGKAPKLLI NO. 50 region: YSASSLYSGVPSRFSGSRSGTDFTLTI SSLQPEDFATYYCQQWAVHSLITF

Fab79H GQGTKVEIKR

SEQ ID Light chain including DIQMTQSPSSLSASVGDRVTITCRASQNGNVRISAVAWYQQKPGKAPKLLI NO. 51 Flag-tag: YSASSLYSGVPSRFSGSRSGTDFTLTI SSLQPEDFATYYCQQWAVHSLITF

Fab79H GQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGSDYKDDDDK

SEQ ID Heavy chain constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH NO. 52 region: TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

KTN0044H; CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHED KTN0117H; PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

SEQ ID Heavy chain variable MGWSCI ILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFNISSY NO. 53 region: SMHWVRQAPGKGLEWVAS IYPYSGYTYYADSVKGRFTI SADTSKNTAYLQM

KTN0044H NSLRAEDTAVYYCARYVYHALDYWGQGTLVTVSS

SEQ ID Heavy chain: MGWSCI ILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFNISSY NO. 54 KTN0044H SMHWVRQAPGKGLEWVAS IYPYSGYTYYADSVKGRFTI SADTSKNTAYLQM

NSLRAEDTAVYYCARYVYHALDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

gccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaag gacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactac gaaaaacataaagtctacgcctgcgaagtcacccatcagggcctgagctcg cccgtcacaaagagcttcaacaggggagagtgtggtggttctgattacaaa gatgacg atgacaaataa

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 73 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCTCTGTTTATTCTATG

Fabl9-S31V CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

TATCCTTATTCTGGCTATACTTATTATGCCGATAGCGTCAAGGGCCGTTTC ACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGC TTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTACGTTTACCAT GCTTTGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 74 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCTCTTCTTATATGATG

Fabl9-S33M CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 75 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCTCTGTTTATATGATG

Fabl9-S31VS33M CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 76 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCGATAGTTATATGATG

Fabl2F CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 77 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCGATGTGTATATGATG

Fabl2G CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 78 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCGATGCGTATATGATG

Fabl2H CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

SEQ ID Heavy chain variable GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCA NO. 79 region DNA sequence: CTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCTCGGTTTATATGATG

Fab 121 CACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATT

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 80 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGATTCGTCATCGTTTGCGTAGG

Fab79A GCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATT

TACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGC CGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGAC TTCGCAACTTATTACTGTCAGCAATGGGCTGTTCATTCTCTGATCACGTTC GGACAGGGTACCAAGGTGGAGATCAAA

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 81 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGATTCGGAAGGTTGCTGTAGCC

Fab79B TGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCA

TCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGG ACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACT TATTACTGTCAGCAATGGGCTGTTCATTCTCTGATCACGTTCGGACAGGGT ACCAAGGTGGAGATCAAA

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 82 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGCGGTATAATACGAGGCCTATG

Fab79C GCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATT

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 83 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGCGTGGTTTGCGTAATGTGGCT

Fab79D GTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTAC

TCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGT TCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTC GCAACTTATTACTGTCAGCAATGGGCTGTTCATTCTCTGATCACGTTCGGA CAGGGT ACCAAGGTGGAGATCAAA

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 84 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGCGGGGGCGTACTGCTGTAGCC

Fab79E TGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCA

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 85 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGCGTGGGGCGAGGAGTGCTGTA

Fab79F GCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCG

GCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCC GGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCA ACTTATTACTGTCAGCAATGGGCTGTTCATTCTCTGATCACGTTCGGACAG GGT ACCAAGGTGGAGATCAAA

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 86 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGCCTTTTAGGCGTGTTGCTGTA

Fab79G GCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCG

SEQ ID Light chain variable GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGAT NO. 87 region DNA sequence: AGGGTCACCATCACCTGCCGTGCCAGTCAGAATGGTAATGTGCGTATTAGT

Fab79H GCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATT

TACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGC CGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGAC TTCGCAACTTATTACTGTCAGCAATGGGCTGTTCATTCTCTGATCACGTTC

GGACAGGGTACCAAGGTGGAGATCAAA

SEQ ID Heavy chain DNA atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtg NO. 88 sequence: cacagcgaggtgcaattggtggagtctggcggtggcctggtgcagccaggg

KTN0044H ggctcactccgtttgtcctgtgcagcttctggcttcaacatctcttcttat tctatgcactgggtgcgtcaggccccgggtaagggcctggaatgggttgca tctatttatccttattctggctatacttattatgccgatagcgtcaagggc cgtttcactataagcgcagacacatccaaaaacacagcctacctacaaatg aacagcttaagagctgaggacactgccgtctattattgtgctcgctacgtt taccatgctttggactactggggtcaaggaaccctggtcaccgtctcctcg gcctccaccaagggtccatcggtcttcccgctagcccccagcagcaagagc accagcggcggcaccgccgccctgggctgcctggtgaaggactacttcccc gagcccgtgaccgtgagctggaacagcggcgccctgaccagcggcgtgcac accttccccgccgtgctgcagagcagcggcctgtacagcctgagcagcgtg gtgaccgtgcccagcagcagcctgggcacccagacctacatctgcaacgtg aaccacaagcccagcaacaccaaggtggacaagaaggtggagcccaagagc tgcgacaagacccacacctgccccccctgccccgcccccgagctgctgggc ggccccagcgtgttcctgttcccccccaagcccaaggacaccctgatgate agcagaacccccgaggtgacctgcgtggtggtggacgtgagccacgaggac cccgaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgcc aagaccaagcccagagaggagcagtacaacagcacctacagagtggtgagc gtgctgaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgc aaggtgagcaacaaggccctgcccgcccccatcgagaagaccatcagcaag gccaagggccagcccagagagccccaggtgtacaccctgccccccagcaga gacgagctgaccaagaaccaggtgagcctgacctgcctggtgaagggcttc taccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaac aactacaagaccaccccccccgtgctggacagcgacggcagcttcttcctg tacagcaagctgaccgtggacaagagcagatggcagcagggcaacgtgttc agctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagc ctgagcctgagccccggcaagtga

SEQ ID Light chain DNA atgggctggagctgcatcatcctgttcctggtggccaccgccaccggtgtg NO. 89 sequence: cacagcgatatccagatgacccagtccccgagctccctgtccgcctctgtg

KTN0044H ggcgatagggtcaccatcacctgccgtgccagtcagtccgtgtccagcgct gtagcctggtatcaacagaaaccaggaaaagctccgaagcttctgatttac tcggcatccagcctctactctggagtcccttctcgcttctctggtagccgt tccgggacggatttcactctgaccatcagcagtctgcagccggaagacttc gcaacttattactgtcagcaatgggctgttcattctctgatcacgttcgga cagggtaccaaggtggagatcaaacgtacggtggccgcccccagcgtgttc atcttcccccccagcgacgagcagctgaagagcggcaccgccagcgtggtg tgcctgctgaacaacttctaccccagagaggccaaggtgcagtggaaggtg gacaacgccctgcagagcggcaacagccaggagagcgtgaccgagcaggac agcaaggacagcacctacagcctgagcagcaccctgaccctgagcaaggcc gactacgagaagcacaaggtgtacgcctgcgaggtgacccaccagggcctg agcagccccgtgaccaagagcttcaacagaggcgagtgctga

SEQ ID Heavy chain DNA atgggctggagctgcatcatcctgttcctggtggccaccgccaccggcgtg NO. 90 sequence: cacagcgaggtgcaattggttgagagcggcggcggcctggtgcagcccggc

KTN0177H ggcagcctgcgcctgagttgcgctgcatctggatttaacatttccgtgtat atgatgcactgggtgcggcaggcaccaggaaagggacttgaatgggtggct tctatctatccctatagcggatacacatattacgccgactcagtcaagggc agatttacgatcagtgctgacacctccaaaaatactgcctatctccaaatg aatagcctcagagccgaagatacggccgtatactattgtgcgcggtacgtg tatcatgctctggactactggggtcagggaactctggtgacagtatcaagt gcctctacaaaaggcccatcagtcttcccgctagcccccagcagcaagagc accagcggcggcaccgccgccctgggctgcctggtgaaggactacttcccc gagcccgtgaccgtgagctggaacagcggcgccctgaccagcggcgtgcac accttccccgccgtgctgcagagcagcggcctgtacagcctgagcagcgtg gtgaccgtgcccagcagcagcctgggcacccagacctacatctgcaacgtg aaccacaagcccagcaacaccaaggtggacaagaaggtggagcccaagagc tgcgacaagacccacacctgccccccctgccccgcccccgagctgctgggc ggccccagcgtgttcctgttcccccccaagcccaaggacaccctgatgate agcagaacccccgaggtgacctgcgtggtggtggacgtgagccacgaggac cccgaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgcc aagaccaagcccagagaggagcagtacaacagcacctacagagtggtgagc gtgctgaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgc aaggtgagcaacaaggccctgcccgcccccatcgagaagaccatcagcaag gccaagggccagcccagagagccccaggtgtacaccctgccccccagcaga gacgagctgaccaagaaccaggtgagcctgacctgcctggtgaagggcttc taccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaac aactacaagaccaccccccccgtgctggacagcgacggcagcttcttcctg tacagcaagctgaccgtggacaagagcagatggcagcagggcaacgtgttc agctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagc ctgagcctgagccccggcaagtga

SEQ ID Light chain DNA atgggctgga gctgcatcat

NO. 91 sequence: cctgttcctggtggccaccgccaccggtgt

KTN0177H gcactccgacattcagatgacccaaagccctagctccctgagcgccagcgt gggggacagggtcactatcacctgtagggctagccagagaggcctgcggaa cgttgccgtggcgtggtaccagcagaagcccggcaaggctcccaagcttct catctattcagcatcatccctctacagcggggtcccatcccgcttttcagg atcccgctccggcactgatttcacactcactatctcttctctgcagcctga ggattttgctacttactactgccaacagtgggctgttcacagtctgattac ctttggccaaggtaccaaggtggagattaagcgtacggtggccgcccccag cgtgttcatcttcccccccagcgacgagcagctgaagagcggcaccgccag cgtggtgtgcctgctgaacaacttctaccccagagaggccaaggtgcagtg gaaggtggacaacgccctgcagagcggcaacagccaggagagcgtgaccga gcaggacagcaaggacagcacctacagcctgagcagcaccctgaccctgag caaggccgactacgagaagcacaaggtgtacgcctgcgaggtgacccacca gggcctgagcagccccgtgaccaagagcttcaacagaggcgagtgctga

Table 4: Antibody and Antibody Fragment CDRs

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. An isolated human anti-KIT antibody, or antigen-binding portion thereof, wherein the antibody, or antigen-binding portion thereof, has one or more of the following biological characteristics:

(a) binds to human KIT with a K_a of at least 1 x 10⁶ M^"1 s^"1;

(b) binds to human KIT with a K_a of at least 1.4 x 10⁶ M^"1 s^"1;

(c) binds to human KIT with a K_a of at least 2.4 x 10⁶ M^"1 s^"1;

(d) binds to human KIT with a K_a of at least 1.4 x 10⁶ M^"1 s^"1;

(e) binds to the D4 domain of human KIT and blocks homotypic interactions between Arg381 and Glu 386;

(f) inhibits SCF-stimulated autophosphorylation of KIT;

(g) inhibits SCF-stimulated autophosphorylation of KIT at a concentration of about 5 nM to about 50 nM;

(h) inhibits SCF-stimulated cell proliferation;

(i) inhibits SCF-stimulated cell proliferation at a concentration of about 5 nM to about

50 nM;

(j) dissociates from human KIT with a Ka of 6.6 x 10^"4 s^"1 or less;

(k) dissociates from human KIT with a Ka of 3.2 x 10^"4 s^"1 or less;

(1) dissociates from human KIT with a Ka of 2.7 x 10^"4 s^"1 or less;

(m) dissociates from human KIT with a Ka of 2.7 x 10^"5 s^"1 or less; or

(n) dissociates from human KIT with a Ka of 1.5 x 10^"5 s^"1 or less.

2. An isolated anti-KIT antibody, or antigen-binding portion thereof, which binds to the same epitope as Fab 19, Fab 121, or Fab79D.

3. An isolated anti-KIT antibody, or antigen-binding portion thereof, which binds to the same epitope as an antibody comprising the six CDRs of Fabl9, Fabl2I or Fab79D.

4. An isolated anti-KIT antibody, or antigen-binding portion thereof, which binds to amino acid residues Pro317-Asn320 of human KIT.

5. The antibody, or antigen-binding portion thereof, of claim 4, which further binds to amino acid residues Glu 329-Asp332, Ile334, Glu336, Lys358, Glu360, Tyr362, Lys364, Glu366, Arg372, Glu376, His378, Thr380 and Arg381 of human KIT.

6. The antibody, or antigen-binding portion thereof, of claim 4, which further binds to amino acid residues Phe316, Val325, Glu329-Asp332, Ile334, Glu336, Glu360, Tyr362- Lys364, Glu366, Arg372, Glu376, His378, Thr380 and Arg381 of human KIT.

7. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising a heavy chain complementary determining region (CDR) 3 which binds to amino acid residues Glu329, Val331, Asp332, Lys358, Glu360, Glu376, His378 and Thr380 of human KIT.

8. The antibody, or antigen-binding portion thereof, of claim 7, which further comprises a heavy chain CDR2 that binds to amino acid residues Pro317, Met318, Asn320, Ile334, Glu336, Lys364 and Arg372 of human KIT.

9. The antibody, or antigen-binding portion thereof, of claim 7, which further comprises a heavy chain CDRl that binds to amino acid residues Ile319, Asn320 and Glu329-Val331 of human KIT.

10. The antibody, or antigen-binding portion thereof, of claim 7, which further comprises a light chain CDR3 that binds to amino acid residues Tyr362, Glu366 and Arg381 of human KIT.

11. The antibody, or antigen-binding portion thereof, of claim 7, which further comprises a light chain CDR2 that binds to amino acid residues Tyr362, Glu366 and Arg381 of human KIT.

12. The antibody, or antigen-binding portion thereof, of claim 7, which further comprises a light chain CDRl that does not bind human KIT.

13. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising a heavy chain complementary determining region (CDR) 3 which binds to amino acid residues Glu329-Asp332, Glu360, Glu376, His378 and Thr380 of human KIT.

14. The antibody, or antigen-binding portion thereof, of claim 13, which further comprises a heavy chain CDR2 that binds amino acids Phe316-Asn320, Ile334, Glu336, Lys364 and Arg372 of human KIT.

15. The antibody, or antigen-binding portion thereof, of claim 13, which further comprises a heavy chain CDRl that binds amino acids Ile319, Asn320, Val325, Asn330 and Glu360 of human KIT.

16. The antibody, or antigen-binding portion thereof, of claim 13, which further comprises a light chain CDR3 that binds amino acids Tyr362 and Glu376 of human KIT.

17. The antibody, or antigen-binding portion thereof, of claim 13, which further comprises a light chain CDR2 that binds amino acids Asn330, Thr380 and Arg381 of human KIT.

18. The antibody, or antigen-binding portion thereof, of claim 13, which further comprises a light chain CDRl that binds amino acids Glu360, Pro363, Lys364 and Glu366 of human KIT.

19. An isolated anti-KIT antibody, or an antigen-binding portion thereof, wherein the human anti-KIT antibody comprises a heavy chain CDR3 domain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO:98.

20. The antibody, or antigen-binding portion thereof, of claim 19, further comprising a heavy chain CDR2 domain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:95, or SEQ ID NO:97.

21. The antibody, or antigen-binding portion thereof, of claim 19, further comprising a heavy chain CDRl domain comprising the amino acid sequence selected from the group consisting of of any one of SEQ ID NOs: 1, 13, 16, 19, 24, 63, 65, 94, 96, 101, 102, 104, 105, 106, 107, 108, 109, 110, 111, and 112.

22. The antibody, or antigen-binding portion thereof, of claim 19, further comprising a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9.

23. The antibody, or antigen-binding portion thereof, of claim 19, further comprising a light chain CDR2 domain comprising the amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 8 and 100.

24. The antibody, or antigen-binding portion thereof, of claim 19, further comprising a light chain CDR1 domain comprising the amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 7, 29, 32, 35, 38, 41, 46, 49, 62, 64, 66, 99, 103, 113, 114, 115, 116, 117, 118, and 119.

25. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising the six CDRs of Fabl9, Fabl2I or Fabs79A-H.

26. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising a heavy chain variable region of any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58.

27. The antibody, or antigen-binding portion thereof, of claim 26, further comprising a light chain variable region of any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60.

28. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising a heavy chain variable region of any one of SEQ ID NOs: 4, 14, 17, 20, 22, 25, 27, 53 or 58 and a light chain variable region of any one of SEQ ID NOs: 10, 30, 33, 36, 39, 42, 44, 47, 50, 55 or 60.

29. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising: a VH CDR1 having the amino acid sequence Χ' 1ΥΧ2ΈΗ, wherein X 1¹ is one or zero amino acid residues, and X is selected from the group consisting of S and M (SEQ ID NO:63);

a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3).

30. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising: a VL CDR1 having the amino acid sequence RASQX¹X²X³X⁴X⁵X⁶X⁷AVA, wherein

31. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising: a VH CDR1 having the amino acid sequence Χ' 1ΥΧ2ΈΗ, wherein X 1¹ is one or zero amino acid residues, and X is selected from the group consisting of S and M (SEQ ID NO:63);

32. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising:

a VH CDR3 comprising the amino acid sequence of YVYHALDY (SEQ ID NO:3).

33. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising:

34. An isolated anti-KIT antibody, or antigen-binding portion thereof, comprising:

X 1 is selected from the group consisting of S, I, R, P and N; X 2 is selected from the group consisting of V, R, Y G and F; X is selected from the group consisting of S, H, K, N, L, R and A; X⁴ is selected from the group consisting of S, R, V and T, X⁵ is selected from the group consisting of no amino acid, L, R, N, S, V and R, X⁶ is selected from the group consisting of no amino acid, R, P, V and I, and X is selected from the group consisting of no amino acid, R, M or S (SEQ ID NO:66); a VL CDR2 having the amino acid sequence SASSLYS (SEQ ID NO: 8); and a VL CDR3 having the amino acid sequence QQWAVHSLIT (SEQ ID NO:9).

35. The antibody, or antigen-binding portion thereof of any one of claims 1-34, wherein the antibody or antigen-binding portion thereof, is selected from the group consisting of a human antibody, a humanized antibody, a bispecific antibody, and a chimeric antibody.

36. The antibody, or antigen-binding portion thereof, of any one of claims 1-34, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain constant region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions.

37. The antibody, or antigen-binding portion thereof, of claim 36, wherein the heavy chain constant region is IgGl.

38. The antibody, or antigen-binding portion thereof, of any one of claims 1-34, which is a Fab fragment, a F(ab')₂ fragment or a single chain Fv fragment.

39. The antibody, or antigen-binding portion thereof, of any one of claims 1-34, wherein the antibody, or antigen-binding portion thereof, binds to the D4 domain of human KIT and blocks homotypic interactions between Arg381 and Glu386.

40. The antibody, or antigen-binding portion thereof of any one of claims 1-39, wherein the antibody, or antigen-binding portion thereof inhibits SCF-stimulated autophosphorylation of KIT.

41. The antibody, or antigen-binding portion thereof of claim 40, wherein the antibody, or antigen-binding portion thereof inhibits SCF-stimulated autophosphorylation of KIT at a concentration of about 5 nM to about 50 nM.

42. The antibody, or antigen-binding portion thereof, of any one of claims 1-39, wherein the antibody, or antigen-binding portion thereof, inhibits SCF-stimulated cell proliferation.

43. The antibody, or antigen-binding portion thereof of claim 42, wherein the antibody, or antigen -binding portion thereof inhibits SCF- stimulated cell proliferation at a concentration of about 5 nM to about 50 nM.

44. The antibody, or antigen-binding portion thereof, of any one of claims 1-43, wherein said antibody, or antigen-binding portion thereof, is conjugated to a different moiety.

45. The antibody, or antigen-binding portion thereof, of claim 44, wherein said moiety is a toxin.

46. The antibody, or antigen-binding portion thereof, of claim 44, wherein said moiety is an anti-cancer agent.

47. A pharmaceutical composition comprising the antibody, or antigen -binding portion thereof, of any one of claims 1-46, and a pharmaceutically acceptable carrier.

48. The pharmaceutical composition of claim 47, further comprising an additional therapeutic agent.

49. A hybridoma which produces the antibody, or antigen binding portion thereof, of any one of claims 1-43.

50. A method of treating or preventing a KIT associated disease in a subject, comprising administering to the subject an effective amount of the antibody, or antigen binding portion thereof, of any one of claims 1-46, thereby treating or preventing the disease in the subject.

51. The method of claim 50, wherein the KIT associated disease is selected from the group consisting of cancer, age-related macular degeneration (AMD), atherosclerosis, rheumatoid arthritis, diabetic retinopathy, and pain associated diseases.

52. The method of claim 51, wherein the cancer is selected from the group consisting of GIST, AML and SCLC.

53. The method of claim 50, wherein the antibody, or antigen-binding portion thereof, is administered in combination with an additional therapeutic agent.

54. A method of inhibiting the phosphorylation of human KIT, the method comprising contacting human KIT with the antibody, or antigen-binding portion thereof, of any one of claims 1-46, thereby inhibiting the phosphorylation of human KIT.

55. A method of inhibiting SCF-stimulated cell proliferation of a cell, the method comprising contacting the cell with the antibody, or antigen-binding portion thereof, of any one of claims 1-46, thereby inhibiting the SCF-stimulated cell proliferation of the cell.

56. A method of inhibiting the interaction between D4 domains of human KIT monomers, the method comprising contacting the human KIT monomers with the antibody, or antigen- binding portion thereof, of any one of claims 1-46, thereby inhibiting the interaction between the D4 domains of the KIT monomers.

57. A method of preventing the dimerization of human KIT monomers, the method comprising contacting a human KIT monomer with the antibody, or antigen-binding portion thereof, of any one of claims 1-46, thereby preventing the dimerization of human KIT monomers.