T CELL RECEPTOR-LIKE ANTIBODIES SPECIFIC FOR A WTI PEPTIDE PRESENTED BY HLA-A2

Abstract

The present invention provides antigen binding proteins that specifically bind to Wilms' tumor protein (WT1), including humanized, chimeric and fully human antibodies against WT1, antibody fragments, chimeric antigen receptors (CARs), fusion proteins, and conjugates thereof. The antigen binding proteins and antibodies bind to HLA-A0201-restricted WT1 peptide. Such antibodies, fragments, fusion proteins and conjugates thereof are useful for the treatment of WT1 associated cancers, including for example, breast cancer, ovarian cancer, prostate cancer, chronic myelocytic leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). In more particular embodiments, the anti-WT1/A antibodies may comprise one or more framework region amino acid substitutions designed to improve protein stability, antibody binding and/or expression levels.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S. application Ser. No. 16/055,535, filed on Aug. 6, 2018, which is a continuation application of U.S. application Ser. No. 15/364,953, filed on Nov. 30, 2016, now U.S. Pat. No. 10,040,865, which is a continuation of U.S. application Ser. No. 14/724,155, filed on May 28, 2015, now U.S. Pat. No. 9,540,448, which is a continuation application of U.S. application Ser. No. 14/008,447 filed Dec. 10, 2013, now U.S. Pat. No. 9,074,000, which is a National Phase filing under 35 U.S.C. .sctn. 371 of PCT International Application PCT/US2012/31892 filed Apr. 2, 2012, and published as WO2012/135854 on Oct. 4, 2012. This application also claims priority to U.S. Provisional Application No. 61/470,635, filed Apr. 1, 2011, and U.S. Provisional Application No. 61/491,392, filed May 31, 2011. These Provisional Applications are hereby incorporated by reference in their entirety into the present disclosure.

SEQUENCE LISTING

[0003] This application contains a Sequence Listing, created on May 28, 2015; the file, in ASCII format, is named 48071A_Seqlisting.txt, and is 80,348 bytes. The file is hereby incorporated by reference in its entirety into the application.

TECHNICAL FIELD

[0004] The present invention relates generally to antibodies against cytosolic proteins. More particularly, the invention relates to antibodies against Wilm's tumor oncogene protein (WT1), specifically antibodies that recognize a WT1 peptide in conjunction with a major histocompatability antigen.

BACKGROUND OF THE INVENTION

[0005] The Wilms' tumor oncogene protein (WT1) is an attractive target for immunotherapy for most leukemias and a wide range of cancers. WT1 is a zinc finger transcription factor that is normally expressed in mesodermal tissues during embryogenesis. In normal adult tissue, WT1 expression is limited to low levels in CD34+ hematopoietic stem cells but is over-expressed in leukemias of multiple lineages and a wide range of solid tumors (1-2). More recently, WT1 expression has been reported to be a marker of minimal residual disease. Increasing transcript levels in patients with acute myeloid leukemia (AML) in morphologic remission have been predictive of overt clinical relapse (3, 4). Furthermore, antibodies to WT1 are detected in patients with hematopoietic malignancies and solid tumors, indicating that WT1 is a highly immunogenic antigen (7).

[0006] For the most part, clinically approved therapeutic monoclonal antibodies (mAbs) recognize structures of cell surface proteins. WT1, however, is a nuclear protein and, therefore, is inaccessible to classical antibody therapy. Up until now, immunotherapy targeting WT1 has been limited to cellular approaches, exclusively aimed at generating WT1-specific cytotoxic CD8 T cell (CTL) responses that recognize peptides presented on the cell surface by MHC class I molecules.

[0007] For induction of CTL responses, intracellular proteins are usually degraded by the proteasome or endo/lysosomes, and the resulting peptide fragments bind to MHC class I or II molecules. These peptide-MHC complexes are displayed at the cell surface where they provide targets for T cell recognition via a peptide-MHC (pMHC)-T cell receptor (TCR) interaction (8, 9). Vaccinations with peptides derived from the WT1 protein induce HLA-restricted cytotoxic CD8 T cells, which are capable of killing tumor cells.

[0008] To improve efficacy, cancer antigens can be targeted with monoclonal antibody therapy. Monoclonal antibody (mAb) therapy has been shown to exert powerful antitumor effects by multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct cell inhibition or apoptosis-inducing effects on tumor cells that over-express the target molecules. Furthermore, mAb can be used as carriers to specifically deliver a cytotoxic moiety such as a radionuclide, cytotoxic drug or toxin to the tumor cells (18).

[0009] A tremendous benefit would exist if, in addition to a cellular immunotherapy approach, a humoral immunotherapy approach was available to target non-cell surface tumor antigens. Therefore, a monoclonal antibody (mAb) that mimics a T cell receptor in that it is specific for a target comprising a fragment of an intracellular protein in conjunction with an MHC molecule, for example, a WT1 peptide/HLA-A2 complex, would be a novel and effective therapeutic agent alone or as a vehicle capable of delivering potent anti-cancer reagents, such as drugs, toxins and radioactive elements. Such mAbs would also be useful as diagnostic or prognostic tools.

SUMMARY OF THE INVENTION

[0010] The present disclosure identifies and characterizes antigen-binding proteins, such as antibodies, that are able to target cytosolic/intracellular proteins, for example, the WT1 oncoprotein. The disclosed antibodies target a peptide/MHC complex as it would typically appear on the surface of a cell following antigen processing of WT1 protein and presentation by the cell. In that regard, the antibodies mimic T-cell receptors in that the antibodies have the ability to specifically recognize and bind to a peptide in an MHC-restricted fashion, that is, when the peptide is bound to an MHC antigen. The peptide/MHC complex recapitulates the antigen as it would typically appear on the surface of a cell following antigen processing and presentation of the WT1 protein to a T-cell.

[0011] The antibodies disclosed specifically recognize and bind to epitopes of a peptide/HLA-A2 complex, particularly a WT1/HLA-A0201 complex. Examples of peptides that are recognized by the antigen-binding proteins of the invention as part of an HLA-peptide complex include, but are not limited to, those shown in Table 7, for example, a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1.)

[0012] In one aspect, therefore, the invention relates to an isolated antibody, or antigen-binding fragment thereof, that binds to a peptide with the amino acid sequence, RMFPNAPYL, when said peptide is bound to an MHC antigen, such as HLA-A2.

[0013] In another aspect, the invention relates to an isolated antigen-binding protein, antibody, or antigen-binding fragment thereof, comprising (A) (i) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; and alight chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; (ii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 20, 21 and 22; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 26, 27 and 28; (iii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 38, 39 and 40; and alight chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences selected from SEQ ID NOS: 44, 45 and 46; (iv) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 62, 63 and 64; (v) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 74, 75 and 76; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 80, 81 and 82; or (vi) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 92, 93 and 94; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 98, 99 and 100.

[0014] In another aspect, the invention relates to an isolated antigen-binding protein, antibody, or antigen-binding fragment thereof, comprising a V.sub.H and V.sub.L comprising first and second amino acid sequences, respectively, selected from SEQ ID NOS: 14 and 16; 32 and 34; 50 and 52; 68 and 70; 86 and 88; and 104 and 106.

[0015] In yet another aspect, the invention relates to an isolated antigen-binding protein, antibody, or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOS: 18, 36, 54, 72, 90, and 108.

[0016] In a related aspect, the isolated antigen-binding protein comprises an antigen-binding region as disclosed in any of Tables 1-8. The antigen-binding protein may be a fusion protein.

[0017] In another aspect, the invention relates to an immunoconjugate comprising a first component which is an antigen-binding protein, antibody or antigen-binding fragment thereof as disclosed herein. The immunoconjugate comprises a second component that is a cytotoxin, a detectable label, a radioisotope, a therapeutic agent, a binding protein or a molecule having a second amino acid sequence. Where the second component is a binding protein or second antibody, the binding protein or second antibody has binding specificity for a target that is different from the HLA-peptide complex for which the first is specific.

[0018] In a related aspect, therefore, the present invention relates to bispecific antibody comprising an antigen-binding protein or functional fragment thereof as described herein.

[0019] In yet another aspect, the invention relates to nucleic acids that encode antigen binding proteins, including antibodies and chimeric antigen receptors specific for a WT1 peptide/HLA complex, in particular the complex of WT1 peptide RMFPNAPYL/HLA-A0201.

[0020] In another related aspect, the invention relates to cells comprising the nucleic acids or antigen binding proteins disclosed herein, including recombinant immune effector cells, such as, T-cells genetically modified to express a chimeric antigen receptor comprising an antigen binding region in accordance with the present disclosure. Cells which have been engineered to produce antibodies in accordance with the disclosure are also encompassed by the invention.

[0021] In a related aspect, the invention relates to vectors comprising the nucleic acids to encode the antigen binding proteins disclosed herein, including vectors to facilitate expression and/or secretion of an antigen binding protein such as an antibody or chimeric antigen receptor in accordance with the present disclosure.

[0022] In a related aspect, the invention relates to pharmaceutical compositions comprising the antigen binding proteins, antibodies, nucleic acids, vectors or cells comprising the nucleic acids or antigen binding proteins disclosed herein, together with a pharmaceutically acceptable carrier.

[0023] In another aspect, the invention relates to a method for detecting WT1 on the surface of cells or tissues using WT1 antibodies of the invention.

[0024] In yet another aspect, the invention relates to methods for treatment of a subject having a WT1-positive disease, comprising administering to the subject a therapeutically effective amount of an antigen binding protein, antibody or antigen binding fragment thereof, nucleic acid encoding the antigen binding protein or antibody or a cell comprising the nucleic acids or proteins as disclosed herein. The WT1-positive disease is a chronic leukemia, acute leukemia or WT1.sup.+ cancer selected from the group consisting of chronic myelocytic leukemia, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma. In some embodiments, the antigen binding protein or antibody is a conjugate thereof having a cytotoxic moiety linked thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows the amino acid sequence of Wilms tumor protein, (GenBank Accession No. P19544) with some HLA-restricted peptides bolded. The 121-140 peptide further encompasses a 9-mer (underlined), RMFPNAPYL (SEQ ID NO: 1), which, in addition to analogs thereof, has been shown to induce WT1-specific cytotoxic T-cell activity.

[0026] FIG. 2 is a graph showing that vaccination with WT1 peptides induces cytotoxic T cells against WT1+ leukemia cells.

[0027] FIG. 3 shows the results of a phage ELISA for specific binding of WT1/A2 (WA) versus PBS control or R3/HLAA0201 (R3).

[0028] FIG. 4 shows specific binding of only WT1 phage antibodies that bind to T2 cells pulsed with WT1A peptide were selected.

[0029] FIG. 5 shows the binding affinity of a full-length IgG1 of a WT1 antibody to RMF/A0201 complex tested by titration of the antibody at various concentrations. Results are shown for T2 cells pulsed with 50 ug/ml RMF (upper panel). Control antibody is shown in the lower panel.

[0030] FIG. 6 shows the dependence on density of RMF/HLA-A0201 complex recognized by WT1 antibody on T2 cells pulsed with RMF (upper panel) or control, RHAMM-R3 (lower panel).

[0031] FIG. 7 shows an expression vector for expression of human antibodies.

[0032] FIG. 8 shows the results of SDS-PAGE analysis of WT1/A2 antibodies under reducing and non-reducing conditions.

[0033] FIG. 9 shows the results of kinetic binding analysis of an WT1/A2 antibody demonstrating affinity of the antibody toward WT1/A2.

[0034] FIG. 10 shows the affinity (K.sub.D) of antibody binding to WT1/A2 complex.

[0035] FIG. 11 shows the mean fluorescence intensity (MFI) by flow cytometry of peptide titration on binding of some embodiments, mAb clone 5 (upper panel), clone 15 (middle panel) and control (lower panel) to live T2 cells pulsed with varying concentrations of peptide, WT1-A, WT1-A1 or control.

[0036] FIG. 12 shows the results of peptide titration on binding of a WT1 antibody, mAb 5 (upper panel), mAb 15 (lower panel) to live T2 cells pulsed with varying concentrations of WT1A peptide.

[0037] FIG. 13 shows the binding specificity of one embodiment, mAb 5, at different concentrations (50 .mu.g/ml upper; 25 .mu.g/ml middle; and 12.5 .mu.g/ml lower) of peptide (R3, WT1-A1, WT1-A or no peptide.)

[0038] FIG. 14 shows the binding specificity of one embodiment, mAb 15, at different concentrations (50 .mu.g/ml upper; 25 .mu.g/ml lower) of peptide (R3, WT1-A1, WT1-A or no peptide).

[0039] FIG. 15 shows dose-dependent binding of mAbs 5 (upper panel) and 15 (lower panel) to T2 cells pulsed with WT1-A, WT1-A1, or RHAMM-R3 peptide.

[0040] FIG. 16 shows binding of mAbs 5 and 15 to U266, a myeloma cell line.

[0041] FIG. 17 shows binding of mAb 15 to BV173, a cell line derived from an individual with (Ph1)-positive acute leukemia.

[0042] FIG. 18 shows the specific binding of ESK1 (#13) to WT1/A2 complex on the surface of T2 cells pulsed with WT1 peptide.

[0043] FIG. 19 and FIG. 20 show that WT1 antibody is able to recognize RMF peptide in which substitution of different positions of the RMF peptide with alanine is made (see also Table 10) and that the loss of binding seen with substitution of position 1 by either alanine (WT1-A1-B) or tyrosine (WT1-A1), was not due to the reduction of peptide binding affinity to the HLA-A2 molecule, as both peptides showed the strongest binding in T2 stabilization assay using the mAb specific for the HLA-A2 molecule, clone BB7.

[0044] FIG. 21 shows recognition by WT1 antibody of naturally presented RMF/HLA-A0201 complex on the cell surface of human mesothelioma cell lines, JMN (WT1.sup.+/A0201.sup.+) but not MSTO (WT1.sup.+/HLA-A0201.sup.-).

[0045] FIG. 22 shows binding of WT1 antibodies to human CML-derived cell line BV173.

[0046] FIG. 23 is a Scatchard analysis based on binding of WT1 antibody to JMN cells and shows an avidity constant of about 0.2 nM.

[0047] FIG. 24 shows WT1 antibody binding to a panel of mesothelioma and leukemia cells.

[0048] FIG. 25 shows the results of flow cytometric analyses gated on CD33 and CD34 double positive AML blast cells from an HLA-A2 positive patient. ESK1 binds to the leukemia blasts.

[0049] FIG. 26 shows the results of flow cytometric analyses gated on CD33 and CD34 double positive AML blast cells from an HLA-A2 negative patient. WT1mAb ESK1 did not bind to the blasts.

[0050] FIG. 27 shows WT1mAb ESK1 mediated ADCC against T2 cells pulsed with RMF peptide.

[0051] FIG. 28 shows the ability of WT1 antibody to mediate ADCC with human effectors in JMN and leukemia cell line BV173 (lower panel) but not MSTO cells.

[0052] FIG. 29 shows that WT1 mAbs are effective against human leukemia cell line BV173 but not HL60 cells, which are not HLA-A2+.

[0053] FIG. 30 shows that WT1 antibody induces ADCC against primary AML blasts from an HLA-A2 positive patient.

[0054] FIG. 31 shows the results of treatment of human BV173 in NSG mice using antibodies of the invention.

[0055] FIG. 32 shows that at later time points, mice treated with WT1 antibody only began to relapse, while antibody with effectors cured 2 of 5 mice.

[0056] FIG. 33 shows that WT1 antibody significantly reduces tumor burden in a dose-dependent manner.

[0057] FIG. 34 shows that antibody with altered carbohydrate in Fc (MAGE) is more active in ADCC than original antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0058] All publications, patents and other references cited herein are incorporated by reference in their entirety into the present disclosure.

[0059] In practicing the present invention, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 3rd edition, J. F. Sambrook and D. W. Russell, ed. Cold Spring Harbor Laboratory Press 2001; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001). The contents of these references and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers' instructions are hereby incorporated by reference as part of the present disclosure. The following abbreviations are used throughout the application:

[0060] Ab: Antibody

[0061] ADCC: Antibody-dependent cellular cytotoxicity

[0062] ALL: Acute lymphocytic leukemia

[0063] AML: Acute myeloid leukemia

[0064] APC: Antigen presenting cell

[0065] .beta.2M: Beta-2-microglobulin

[0066] BiTE: Bi-specific T cell engaging antibody

[0067] CAR: Chimeric antigen receptor

[0068] CDC: Complement dependent cytotoxicity

[0069] CMC: Complement mediated cytotoxicity

[0070] CDR: Complementarity determining region (see also HVR below)

[0071] C.sub.L: Constant domain of the light chain

[0072] CH.sub.1: 1.sup.st constant domain of the heavy chain

[0073] CH.sub.1, 2, 3: 1.sup.st, 2.sup.nd and 3.sup.rd constant domains of the heavy chain

[0074] CH.sub.2, 3: 2.sup.nd and 3.sup.rd constant domains of the heavy chain

[0075] CHO: Chinese hamster ovary

[0076] CTL: Cytotoxic T cell

[0077] E:T Ratio: Effector:Target ratio

[0078] Fab: Antibody binding fragment

[0079] FACS: Flow assisted cytometric cell sorting

[0080] FBS: Fetal bovine serum

[0081] FR: Framework region

[0082] HC: Heavy chain

[0083] HLA: Human leukocyte antigen

[0084] HVR-H: Hypervariable region-heavy chain (see also CDR)

[0085] HVR-L: Hypervariable region-light chain (see also CDR)

[0086] Ig: Immunoglobulin

[0087] IRES: Internal ribosome entry site

[0088] K.sub.D: Dissociation constant

[0089] k.sub.off: Dissociation rate

[0090] k.sub.on: Association rate

[0091] MHC: Major histocompatibility complex

[0092] MM: Multiple myeloma

[0093] scFv: Single-chain variable fragment

[0094] TCR: T cell receptor

[0095] V.sub.H: Variable heavy chain includes heavy chain hypervariable region and heavy chain variable framework region

[0096] V.sub.L: Variable light chain includes light chain hypervariable region and light chain variable framework region

[0097] WT1: Wilms tumor protein 1

[0098] In the description that follows, certain conventions will be followed as regards the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art.

[0099] An "antigen-binding protein" is a protein or polypeptide that comprises an antigen-binding region or antigen-binding portion, that is, has a strong affinity to another molecule to which it binds. Antigen-binding proteins encompass antibodies, chimeric antigen receptors (CARs) and fusion proteins.

[0100] "Antibody" and "antibodies" as those terms are known in the art refer to antigen binding proteins of the immune system. The term "antibody" as referred to herein includes whole, full length antibodies having an antigen-binding region, and any fragment thereof in which the "antigen-binding portion" or "antigen-binding region" is retained, or single chains, for example, single chain variable fragment (scFv), thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V.sub.H) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V.sub.L) and alight chain constant C.sub.L region. The light chain constant region is comprised of one domain, C.sub.L. The V.sub.H and V.sub.L 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 V.sub.H and V.sub.L 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. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0101] The term "antigen-binding portion" or "antigen-binding region" of an antibody, as used herein, refers to that region or portion of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an peptide/HLA complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody fragments" of an antibody include a Fab fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H, C.sub.L and CH1 domains; a F(ab).sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V.sub.H and CH1 domains; a Fv fragment consisting of the V.sub.L and V.sub.H domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a V.sub.H domain; and an isolated complementarity determining region (CDR).

[0102] Furthermore, although the two domains of the Fv fragment, V.sub.L and V.sub.H, 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 V.sub.L and V.sub.H regions pair to form monovalent molecules. These are 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. 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0103] An "isolated antibody" or "isolated antigen-binding protein" is one which has been identified and separated and/or recovered from a component of its natural environment. "Synthetic antibodies" or "recombinant antibodies" are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art.

[0104] Traditionally, the MHC-peptide complex could only be recognized by a T-cell receptor (TCR), limiting our ability to detect an epitope of interest using T cell-based readout assays. In the present disclosure, antigen binding proteins, including antibodies, having an antigen-binding region based on scFvs that are selected from human scFv phage display libraries using recombinant HLA-peptide complexes are described. These molecules demonstrated exquisite specificity, for example as shown with anti-WT1 antibodies that recognize only HLA-A2-RMFPNAPYL complexes. In addition, along with their inability to bind to HLA-complexes containing other peptides, the molecules were also unable to bind to the peptides themselves, further demonstrating their TCR-like specificity.

[0105] The scFvs of the disclosure selected by phage display were initially tested for their ability to bind to peptide presented on the surface of HLA-positive cells. After T2 cells were incubated in the presence of peptide, fluorescently labeled antibodies could be used to selectively recognize the antigen pulsed cells using flow cytometry.

[0106] In some embodiments, the invention includes antibodies that have the scFv sequence fused to one or more constant domains of the heavy to form an antibody with an Fc region of a human immunoglobulin to yield a bivalent protein, increasing the overall avidity and stability of the antibody. In addition, the Fc portion allows the direct conjugation of other molecules, including but not limited to fluorescent dyes, cytotoxins, radioisotopes etc. to the antibody for example, for use in antigen quantitation studies, to immobilize the antibody for affinity measurements, for targeted delivery of a therapeutic agent, to test for Fc-mediated cytotoxicity using immune effector cells and many other applications.

[0107] The results presented here highlight the specificity, sensitivity and utility of the antibodies of the invention in targeting MHC-peptide complexes.

[0108] The molecules of the invention are based on the identification and selection of single chain variable fragments (scFv) using phage display, the amino acid sequence of which confers the molecules' specificity for the MHC restricted peptide of interest and forms the basis of all antigen binding proteins of the disclosure. The scFv, therefore, can be used to design a diverse array of "antibody" molecules, including, for example, full length antibodies, fragments thereof, such as Fab and F(ab').sub.2, minibodies, fusion proteins, including scFv-Fc fusions, multivalent antibodies, that is, antibodies that have more than one specificity for the same antigen or different antigens, for example, bispecific T-cell engaging antibodies (BiTe), tribodies, etc. (see Cuesta et al., Multivalent antibodies: when design surpasses evolution. Trends in Biotechnology 28:355-362 2010).

[0109] In an embodiment in which the antigen-binding protein is a full length antibody, the heavy and light chains of an antibody of the invention may be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or may include an antigen-binding portion (a Fab, F(ab').sub.2, Fv or a single chain Fv fragment ("scFv")). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the immunoglobulin isotype is selected from IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.

[0110] In constructing a recombinant immunoglobulin, appropriate amino acid sequences for constant regions of various immunoglobulin isotypes and methods for the production of a wide array of antibodies are known to those of skill in the art.

[0111] In one embodiment, the antibody or other antigen binding protein is an anti-WT1/HLA-A2 scFv or or antigen-binding fragment thereof having an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 18 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In some embodiments, the anti-WT1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 1.

TABLE-US-00001 TABLE 1 Antigen WT1 (Ext002 #3) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 1 2 3 VH GGTFSSYAIS GIIPIFGTANYAQKFQG RIPPYYGMDV (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) DNA ggaggcaccttcagcag gggatcatccctatctttggtac cggattcccccgtactacggtat ctatgctatcagc agcaaactacgcacagaagtt ggacgtc (SEQ ID NO: 7) (SEQ ID NO: 5) ccagggc (SEQ ID NO: 6) VL SGSSSNIGSNYVY RSNQRPS AAWDDSLNGVV (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) DNA tctggaagcagctccaac aggagtaatcagcggccctca gcagcatgggatgacagcctg atcggaagtaattatgtat (SEQ ID NO: 12) aatggtgtggta ac (SEQ ID NO: 11) (SEQ ID NO: 13) Full QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE VH WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY CARRIPPYYGMDVWGQGTTVTVSS (SEQ ID NO: 14) DNA caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg cagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctgg ggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 15) Full QTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL VL LIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD SLNGVVFGGGTKLTVLG (SEQ ID NO: 16) DNA cagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgtt ctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcc cccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctcca agtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgt gcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg t (SEQ ID NO: 17) scFv QTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL LIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD SLNGVVFGGGTKLTVLGSRGGGGSGGGGSGGGSLEMAQVQLVQSG AEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFG TANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRIPPYY GMDVWGQGTTVTVSS (SEQ ID NO: 18) DNA cagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgtt ctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcc cccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctcca agtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgt gcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg ttctagaggtggtggtggtagcggcggcggcggctctggtggtggatccctcgagatggc ccaggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctg caaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaag ggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagg gcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctga gatctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctg gggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 19)

[0112] In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen-binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 36 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 2.

TABLE-US-00002 TABLE 2 Antigen WT1 (Ext002 #5) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 1 2 3 VH GDSVSSNSAAWN RTYYGSKWYNDYAVS GRLGDAFDI (SEQ ID NO: 20) VKS (SEQ ID NO: 21) (SEQ ID NO: 22) DNA ggggacagtgtctctagc aggacatactacgggtccaag ggtcgcttaggggatgcttttga aacagtgctgcttggaac tggtataatgattatgcagtatct tatc (SEQ ID NO: 25) (SEQ ID NO: 23) gtgaaaagt (SEQ ID NO: 24) VL RASQSISSYLN AASSLQS QQSYSTPLT (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28) DNA cgggcaagtcagagcatt gctgcatccagtttgcaaagt caacagagttacagtacccct agcagctatttaaat (SEQ ID NO: 30) ctcact (SEQ ID NO: 31) (SEQ ID NO: 29) Full QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGL VH EWLGRTYYGSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTA VYYCARGRLGDAFDIWGQGTMVTVSS (SEQ ID NO: 32) DNA caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgt gccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcg agaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctg tgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactct gtgactcccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctgg ggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 33) Full DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY VL AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT FGGGTKVDIKR (SEQ ID NO: 34) DNA gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgt (SEQ ID NO: 35) scFv DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT FGGGTKVDIKRSRGGGGSGGGGSGGGGSLEMAQVQLQQSGPGLVK PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWY NDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGRLGDAF DIWGQGTMVTVSS (SEQ ID NO: 36) DNA gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgttctagaggtg gtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtac agctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatct ccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggc cttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctgtgaaaa gtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactc ccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctggggccaa gggacaatggtcaccgtctcttca (SEQ ID NO: 37)

[0113] In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 54 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 3.

TABLE-US-00003 TABLE 3 Antigen WT1 (Ext002 #13) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 1 2 3 VH GYSFTNFWIS RVDPGYSYSTYSPSF VQYSGYYDWFDP (SEQ ID NO: 38) QG (SEQ ID NO: 39) (SEQ ID NO: 40) DNA ggatacagcttcaccaact agggttgatcctggctactctta gtacaatatagtggctactatg tctggatcagc tagcacctacagcccgtccttc actggttcgacccc (SEQ ID NO: 41) caaggc (SEQ ID NO: 42) (SEQ ID NO: 43) VL SGSSSNIGSNTVN SNNQRPS AAWDDSLNGWV (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46) DNA tctggaagcagctccaac agtaataatcagcggccctca gcagcatgggatgacagcct atcggaagtaatactgtaa (SEQ ID NO: 48) gaatggttgggtg ac (SEQ ID NO: 47) (SEQ ID NO: 49) Full VH QMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLE WMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTA MYYCARVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 50) DNA cagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatctcct gtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaa ggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttccaag gccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaag gcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacc cctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 51) Full QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK VL LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGWVFGGGTKLTVLG (SEQ ID NO: 52) DNA caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggt (SEQ ID NO: 53) scFv QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGWVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQMQLV QSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLEWMGR VDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCA RVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 54) DNA caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatgg cccagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatct cctgtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccggga aaggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttcca aggccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctga aggcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcga cccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 55)

[0114] In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 72 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO:1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 4.

TABLE-US-00004 TABLE 4 Antigen WT1 (Ext002 #15) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 1 2 3 VH GYNFSNKWIG IIYPGYSDITYSPSFQG HTALAGFDY (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ ID NO: 58) DNA ggctacaactttagcaaca atcatctatcccggttactcgga cacacagctttggccggctttg agtggatcggc catcacctacagcccgtccttc actac (SEQ ID NO: 61) (SEQ ID NO: 59) caaggc (SEQ ID NO: 60) VL RASQNINKWLA KASSLES QQYNSYAT (SEQ ID NO: 62) (SEQ ID NO: 63) (SEQ ID NO: 64) DNA Cgggccagtcagaatatc aaggcgtctagtttagaaagt caacaatataatagttatgcga aataagtggctggcc (SEQ ID NO: 66) cg (SEQ ID NO: 67) (SEQ ID NO: 65) Full QVQLVQSGAEVKKPGESLKISCKGSGYNFSNKWIGWVRQLPGRGLE VH WIAIIYPGYSDITYSPSFQGRVTISADTSINTAYLHWHSLKASDTAMYYC VRHTALAGFDYWGLGTLVTVSS (SEQ ID NO: 68) DNA caggtgcagctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctg taagggttctggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagagg cctggagtggatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggc cgcgtcaccatctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggc ctcggacaccgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgg gcaccctggtcaccgtctcctca (SEQ ID NO: 69) Full DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI VL YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT FGQGTKVEIKR (SEQ ID NO: 70) DNA gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO: 71) scFv DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT FGQGTKVEIKRSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKK PGESLKISCKGSGYNFSNKWIGWVRQLPGRGLEWIAI IYPGYSDITYSP SFQGRVTISADTSINTAYLHWHSLKASDTAMYYCVRHTALAG FDYWGL GTLVTVSS (SEQ ID NO: 72) DNA gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgttctagaggtggt ggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtgcag ctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctgtaagggttct ggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagaggcctggagtg gatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggccgcgtcacc atctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggcctcggacac cgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgggcaccctggt caccgtctcctca (SEQ ID NO: 73)

[0115] In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 90 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 5.

TABLE-US-00005 TABLE 5 Antigen WT1 (Ext002 #18) Peptide RMFPNAPYL (SEQ ID NO: 1) CDRs: 1 2 3 VH GFTFDDYGMS GINWNGGSTGYADS ERGYGYHDPHDY (SEQ ID NO: 74) VRG (SEQ ID NO: 75) (SEQ ID NO: 76) DNA gggttcacctttgatgattat ggtattaattggaatggtggt gagcgtggctacgggtacca ggcatgagc agcacaggttatgcagactc tgatccccatgactac (SEQ ID NO: 77) tgtgaggggc (SEQ ID (SEQ ID NO: 79) NO: 78) VL GRNNIGSKSVH DDSDRPS QVWDSSSDHVV (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82) DNA gggagaaacaacattgg gatgatagcgaccggccctc caggtgtgggatagtagtagt aagtaaaagtgtgcac a (SEQ ID NO: 84) gatcatgtggta (SEQ ID NO: 83) (SEQ ID NO: 85) Full EVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKG VH LEWVSGINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAE DTALYYCARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 86) DNA gaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtccctgagactctcct gtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagctccagggaag gggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgagg ggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctg agagccgaggacacggccttgtattactgtgcgagagagcgtggctacgggtaccatgatccc catgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 87) Full QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL VL VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVLG (SEQ ID NO: 88) DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct gaccgtcctaggt (SEQ ID NO: 89) scFv QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVLGSRGGGGSGGGGSGGSLEMAEVQLVQ SGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVS GINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTALYY CARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 90) DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct gaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggatccc tcgagatggccgaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtcc ctgagactctcctgtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaag ctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgca gactctgtgaggggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaa atgaacagtctgagagccgaggacacggccttgtattactgtgcgagagagcgtggctacggg taccatgatccccatgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 91)

[0116] In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 108 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 6.

TABLE-US-00006 TABLE 6 Antigen WT1 (Ext002 #23) Peptide RMFPNAPYL (SEQ ID NO. 1) CDRs: 1 2 3 VH GFSVSGTYMG LLYSGGGTYHPASLQ GGAGGGHFDS (SEQ ID NO. 92) G (SEQ ID NO. 93) (SEQ ID NO. 94) DNA gggttctccgtcagtggcac cttctttatagtggtggcggcac gaggggcaggaggtggcc ctacatgggc (SEQ ID ataccacccagcgtccctgca actttgactcc (SEQ ID NO. 95) gggc (SEQ ID NO. 96) NO. 97) VL TGSSSNIGAGYDVH GNSNRPS AAWDDSLNGYV (SEQ ID NO. 98) (SEQ ID NO. 99) (SEQ ID NO. 100) DNA actgggagcagctccaac ggtaacagcaatcggccctca gcagcatgggatgacagcct atcggggcaggttatgatgt (SEQ ID NO. 102) gaatggttatgtc acac (SEQ ID NO. 101) (SEQ ID NO. 103) Full EVQLVETGGGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLE VH WVALLYSGGGTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVY YCAKGGAGGGHFDSWGQGTLVTVSS (SEQ ID NO. 104) DNA gaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcagactctcctg tgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagggaaggg actggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgcagggccg attcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaagccgag gacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctggggcca aggcaccctggtgaccgtctcctca (SEQ ID NO. 105) Full QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK VL LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTKLTVLG (SEQ ID NO. 106) DNA cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta ggt (SEQ ID NO. 107) scFv QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVETG GGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLEWVALLYSGG GTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVYYCAKGGAGG GHFDSWGQGTLVTVSS (SEQ ID NO. 108) DNA cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta ggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag atggccgaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcaga ctctcctgtgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagg gaagggactggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgca gggccgattcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaa gccgaggacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctg gggccaaggcaccctggtgaccgtctcctca (SEQ ID NO. 109)

[0117] Other embodiments of the disclosed antibodies and antigen binding proteins encompass those comprising light and heavy hypervariable regions and constant regions, for example as shown in Tables 7 (heavy chain), 8 (light chain) and 9 (constant regions).

TABLE-US-00007 TABLE 7 CDR-H1 CDR-H2 CDR-H3 SEQ ID NO. Group I EXT002-12(166) SNAVAWN RTYRGSTYY---ALSV G-SNSAFDF 119-121 EXT002-5(184) SNSAAWN RTYYGSKWYNDYAVSV GRLGDAFDI 122-124 EXT002-8(184) SDGAAWN RTYYRSKWYNDYAVSV GDYYYGMDV 125-127 Consensus(191) SNAAAWN RTYYGSKWYNDYAVSV GAFDI 128-130 Group II EXT002-14(163) SYWIS RIDPSDSYTNYSPSFQG GD------YDFYLDP-- 131-133 EXT002-25(163) SYGIS WISAYNGNTNYAQKLQG DLYSSGWYESYYYGMDV 134-136 EXT002-3(186) SYAIS GIIPIFGTANYAQKFQG RIP-P------YYGMDV 137-139 EXT002-30(163) SYGIS WISAHNGNTNYAQKLQG DR-------VWFGDLSD 134, 140, 141 EXT002-33(163) SYAIS GIIPIFGTANYAQKEQG NYDFWSG-----DAFDI 137, 142, 143 Consensus(188) SYAIS IPGTNYAQKFQG FYGMDV 137, 144, 145 Group III EXT002-34(161) DYGMS GINWNGGSTGYADSV ERGY-GYHDPHDY 146-148 EXT002-40(157) NYTMN SISLSGAYIYYADSL EGYSSSVYDAFDL 149-151 EXT002-45(165) SYGMH GILSDGGKDYYVDSV CSSN-YGNDAFDI 152-154 EXT002-48(165) TYSMN SISSGAYSIFYADSV DQYYGDKWDAFDI 155-157 Consensus(170) SYGMN SISSGGSIYYADSV EYYWDAFDI 158-160

TABLE-US-00008 TABLE 8 CDR-L1 CDR-L2 CDR-L3 SEQ ID NOS. Group I EXT002-1(46) CSGSSSNIGS-NTVN SNNQRPSG AAWDDSLNG--WVFG 161-163 EXT002-10(46) CSGSSSNIGS-NTVN SNNQRPSG EAWDDSLKG--PVFG 161, 162, 164 EXT002-12(22) CTGSSSNIGAGYDVH GNSNRPSG QSYDSSLSADNYVFG 165-167 EXT002-13(46) CSGSSSNIGS-NTVN SNNQRPSG AAWDDSLNG--WVFG 161-163 EXT002-2(46) CSGSSSNIGR-NIVN SNIERPSG ASWDDSLNG--VLFG 168-170 EXT002-20(46) CSGSRSNIAS-NGVG KNDQRPSG SAWDDSLDGH-VVFG 171-173 EXT002-23(46) CTGSSSNIGAGYDVH GNSNRPSG AAWDDSLNG--YVFG 165, 166, 174 EXT002-25(22) CSGSSSNIGS-STVN SNSQRPSG AAWDDSLNG--VVFG 175-177 EXT002-3(46) CSGSSSNIGS-NYVY RSNQRPSG AAWDDSLNG--VVFG 178, 179, 177 EXT002-30(22) CSGSSSNIGR-NTVN SNNQRPSG AAWDDSLNG--YVFG 180, 162, 174 EXT002-33(22) CSGSSSNIGN-DYVS DNNKRPSG GTWDNSLSA--WVFG 181-183 EXT002-36(22) CSGSSSNIGS-NSVY NNNQRPSG ATWDDSLSG--WVFG 184-186 EXT002-40(22) CSGSSSNIGS-NYVY RNNQRPSG AAWDDSLSA--WVFG 178, 187, 188 EXT002-42(46) CSGSTSNIGS-YYVS DNNNRPSG GTWDSSLSA--WVFG 189-191 EXT002-45(22) CSGSSSNIGN-NYVS DNNKRPSG GTWDSSLSA--WVFG 192, 182, 191 EXT002-48(22) CSGSNSNIGT-NTVT SNFERPSG SAWDDSFNG--PVFG 193-195 EXT002-6(46) CSGSSSNIGS-NYVS RNNQRPSG AAWDDGLRG--YVFG 196, 187, 197 EXT002-9(22) CSGSSSNIGS-NTVN SNNQRPSG EAWDDSLKG--PVFG 161, 162, 164 Consensus(46) CSGSSSNIGSNV NNQRPSG AAWDDSLGWVFG 161-163 Group II EXT002-24(24) RASQSISSYLN AASSLQS QQSYSTP--T 198-200 EXT002-31(24) RASQGISNYLA AASTLQS QKYNSAPGVT 201-203 EXT002-35(24) RASQSINGWLA RASTLQS QQSSSLP-FT 204-206 EXT002-5(48) RASQSISSYLN AASSLQS QQSYSTP-LT 198-200 EXT002-7(48) RASQGISYYLA AASTLKS QQLNSYP-LT 207-209 EXT002-B(48) RASQSISSYLN AASSLQS QQSYSTP-WT 198-200 Consensus(48) RASQSISSYLN AASSLQS QQSYSTPLT 198-200 Group III EXT002-16(23) GGNNIGSKSVH DDSDRPS QVWDSSSDHPV 210-212 EXT002-17(47) GGNNIGSKSVH DDSDRPS QVWDSSGDHPV 210, 211, 213 EXT002-19(47) GGNNIGSKSVH YDSDRPS QVWDSSSDHPV 210, 214, 212 EXT002-21(19) GGTNIGSRFVH DDSDRPS QVWDSSGDHPV 215, 211, 213 EXT002-22(47) GGNNVESKSVH YDRDRPS EVWDSGSDHPV 216-218 EXT002-32(23) GGKNIGSKSVH YDSDRPS QVWDSGSDHYV 219, 214, 220 EXT002-34(23) GGNNIGSKSVH DDSDRPS QVWISSGDRVI 210, 211, 221 EXT002-43(23) GGDNIGSQGVH YDTDRPS QVWGASSDHPV 222-224 Consensus(47) GGNNIGSKSVH YDSDRPS QVWDSSSDHPV 210, 214, 212 Group IV EXT002-11(47) TGTSSDVGGYNYVS DVSKRPS GIYTYSDSW--V 225-227 EXT002-14(23) TGTSSDVGGYNYVS DVGNRPS SSYTSSSTR--V 225, 228, 229 EXT002-26(23) TGTRSDVGLYNYVA DVIYRPG SSYTNIGTV--L 230-232 EXT002-4(47) TGTSSDFGDYDYVS DVSDRPS QSYDSSLSGSGV 233-235 Consensus(47) TGTSSDVGGYNYVS DVSRPS SSYTSSSV 225, 234, 229

TABLE-US-00009 TABLE 9 Constant Regions Human heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL constant region TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT and IgG1 Fc domain KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR sequence TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO. 236) Human light chain TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL (kappa) QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO. 237) Human light chain QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGS (lambda) PVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG STVEKTVAPTECS (SEQ ID NO. 238)

[0118] The invention relates to recombinant antigen-binding proteins, antibodies and antigen binding fragments thereof that specifically recognize epitopes of a complex of a peptide/protein fragment derived from an intracellular protein, and an MHC class I molecule, for example, as the complex might be displayed at the cell surface during antigen presentation. The heavy and light chains of an antibody of the invention may be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or may include an antigen-binding portion (a Fab, F(ab').sub.2, Fv or a single chain Fv fragment ("scFv")). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.

[0119] The antibodies and antigen binding proteins of the present invention are intended to encompass bispecific antibodies, including bispecific T-cell engaging antibodies, that is, antibodies comprising two antibody variable domains on a single polypeptide chain that are able to bind two separate antigens. Where a first portion of a bispecific antibody binds an antigen on a tumor cell for example and a second portion of a bispecific antibody recognizes an antigen on the surface of a human immune effector cell, the antibody is capable of recruiting the activity of that effector cell by specifically binding to the effector antigen on the human immune effector cell. In some instances, bispecific antibodies, therefore, are able to form a link between effector cells, for example, T cells and tumor cells, thereby enhancing effector function.

[0120] In one embodiment, the constant region/framework region is altered, for example, by amino acid substitution, to modify the properties of the antibody (e.g., to increase or decrease one or more of: antigen binding affinity, Fc receptor binding, antibody carbohydrate, for example, glycosylation, fucosylation etc., the number of cysteine residues, effector cell function, effector cell function, complement function or introduction of a conjugation site). Furthermore, conjugation of the antibody to a drug, toxin, radioisotope, cytokine, inflammatory peptide or cytotoxic agent is also contemplated.

[0121] In one embodiment, the antibody is an anti-WT1/A2 antibody and comprises the human IgG1 constant region and Fc domain shown in Table 9. In one embodiment, the anti-WT1/A2 antibody comprises a human kappa sequence, or a human lambda sequence having the sequence set forth in Table 9. The amino acid sequences for some complementarity determining regions (CDRs) of antibodies of the invention are shown in Tables 1-8.

[0122] The present invention is based on the identification of antigen-specific binding sequences from which a variety of antigen-binding proteins can be produced. In addition to antibodies specific for an antigen that represents a protein fragment (peptide)/HLA complex similar to that typically recognized by a T-cell receptor following antigen processing and presentation of the protein to the T-cell, identification of amino acid and nucleic sequences as disclosed herein for the preparation of antibodies can also be used to generate other antigen-binding molecules including chimeric antigen receptors (CARs), with specificity for the protein fragment (peptide)/HLA complex. These can be incorporated into cells to make them specifically ctyotoxic to the antigen expressing cell.

[0123] The present invention employs a novel approach to obtaining therapeutic antibodies to any protein, including those proteins that are inaccessible because they are not expressed on the cell surface. Nearly any intracytoplasmic or intranuclear protein (in addition to cell surface proteins) is a potential target for the approach described herein. This includes, but is not limited to, oncogenic proteins, transcription factors, enzymes, etc.

[0124] In order to target tumor antigens derived from intracellular or nuclear proteins, development of a therapeutic antibody an uncommon approach was required. This approach is to generate recombinant mAbs that recognize the peptide/MHC complex expressed on the cell surface, with the same specificity as a T-cell receptor (TCR). Such mAbs share functional homology with TCRs regarding target recognition, but confer higher affinity and capabilities of arming with potent cytotoxic agents that antibodies feature. Technically, TCR-like mAbs may be generated by conventional hybridoma techniques known to those of skill in the art, to produce human, humanized or chimeric antibodies.

[0125] Furthermore, fully-human mAbs are preferred for therapeutic use in humans because murine antibodies cause an immunogenicity reaction, known as the HAMA (human anti-mouse antibodies) response (24, 25), when administered to humans, causing serious side effects, including anaphylaxis and hypersensitivity reactions. This immunogenicity reaction is triggered by the human immune system recognizing the murine antibodies as foreign because of slightly different amino acid sequences from natural human antibodies. Humanization methods known in the art (26, 27) can be employed to reduce the immunogenicity of murine-derived antibodies (28).

[0126] Recently, the use of phage display libraries has made it possible to select large numbers of Ab repertoires for unique and rare Abs against very defined epitopes (for more details on phage display see McCafferty et al., Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348: 552-554.) The rapid identification of human Fab or single chain Fv (ScFV) fragments highly specific for tumor antigen-derived peptide-MHC complex molecules has thus become possible (19-22). More recently, immuno-toxins, generated by fusing TCR-like Fab specific for melanoma Ag MART-1 26-35/A2 or gp100 280-288/A2 to a truncated form of Pseudomonas endotoxin, have been shown to inhibit human melanoma growth both in vitro and in vivo (23). In addition, by engineering full-length mAb using the Fab fragments, it is possible to directly generate a therapeutic human mAb, by-passing months of time-consuming work, normally needed for developing therapeutic mAbs. The present invention involves the development of a TCR-like, fully human mAb that recognizes, for example, the WT1 peptide/HLA-A2 complex (RMFPNAPYL, SEQ ID NO: 1) for cancer therapy.

[0127] Recombinant antibodies with TCR-like specificity represent a new and valuable tool for research and therapeutic applications in tumor immunology and immunotherapy. WT1 is a well-established and validated tumor antigen that has been investigated throughout the world as a marker, prognostic factor and therapeutic target. It was recently prioritized as the top priority tumor antigen by an NCI task force (29).

Identification of Peptides with High Predictive Binding to HLA Molecules

[0128] In one embodiment, the present invention relates to a method for the generation of antibodies that specifically bind to HLA-restricted peptides, which, when presented as part of a peptide/MHC complex are able to elicit a specific cytotoxic T-cell response. HLA class I molecules present endogenous derived peptides of about 8-12 amino acids in length to CD8+ cytotoxic T lymphocytes. Peptides to be used in the method of the present invention are generally about 6-22 amino acids in length, and in some embodiments, between about 9 and 20 amino acids and comprise an amino acid sequence derived from a protein of interest, for example, human WT1 protein (Genbank accession no. P19544) or an analog thereof.

[0129] Peptides suitable for use in generating antibodies in accordance with the method of the present invention can be determined based on the presence of HLA-A0201-binding motifs and the cleavage sites for proteasomes and immune-proteasomes using computer prediction models known to those of skill in the art. For predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007)

[0130] HLA-A*0201 is expressed in 39-46% of all caucasians and therefore, represents a suitable choice of MHC antigen for use in the present method. For preparation of one embodiment of a WT1 peptide antigen, amino acid sequences and predicted binding of putative CD84+ epitopes to HLA-A0201 molecules were identified using the predictive algorithm of the SYFPEITHI database (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007).

[0131] Once appropriate peptides have been identified, peptide synthesis may be done in accordance with protocols well known to those of skill in the art. Because of their relatively small size, the peptides of the invention may be directly synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. The synthesis of peptides in solution phase has become a well-established procedure for large-scale production of synthetic peptides and as such is a suitable alternative method for preparing the peptides of the invention. (See for example, Solid Phase Peptide Synthesis by John Morrow Stewart and Martin et al. Application of Almez-mediated Amidation Reactions to Solution Phase Peptide Synthesis, Tetrahedron Letters Vol. 39, pages 1517-1520 1998.)

[0132] Each of the peptides used in the protocols described herein was purchased and synthesized by Genemed Synthesis, Inc. (San Antonio, Tex.) using fluorenylmethoxycarbonyl chemistry and solid-phase synthesis and purified by high-pressure liquid chromatography. The quality of the peptides was assessed by high-performance liquid chromatography analysis, and the expected molecular weight was observed using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and 70% to 90% pure. The peptides were dissolved in DMSO and diluted in PBS (pH 7.4) or saline at 5 mg/mL and stored at -80.degree. C.

[0133] Subsequent to peptide selection, binding activity of selected peptides is tested using the antigen-processing-deficient T2 cell line, which increases expression of HLA-A when stabilized by a peptide in the antigen-presenting groove. Briefly, T2 cells are pulsed with peptide for a time sufficient to induce HLA-A expression. HLA-A expression of T2 cells is then measured by immunostaining with a fluorescently labeled monoclonal antibody specific for HLA-A (for example, BB7.2) and flow cytometry. Fluorescence index (FI) is calculated as the mean fluorescence intensity (MFI) of HLA-A0201 on T2 cells as determined by fluorescence-activated cell-sorting analysis, using the formula FI=(MFI[T2 cells with peptide]/MFI [T2 cells without peptide]-1.

[0134] Fully human T-cell receptor (TCR)-like antibodies to Wilm's tumor oncognene protein (WT1) were produced using the method disclosed herein. TCR-like anti-WT1 antibodies generated by phage display technology are specific for a WT1 peptide/HLA complex similar to that which induces HLA-restricted cytotoxic CD8 T-cells.

[0135] The WT1 protein sequence was screened using the SYFPEITHI algorithm and WT1 peptides (for example, peptides designated 428, 328, and 122) were identified that had predicted high-affinity binding to multiple HLA molecules that are highly expressed in the Caucasian population. Peptide 428 spans WT1 amino acids 428-459, peptide 328 spans WT1 amino acids 328-349, and peptide 122 spans WT1 amino acids 122-140 (see FIG. 1)

[0136] Heteroclitic peptides can also be designed by conservative amino acid substitutions of MHC-binding residues expected to enhance the affinity toward the MHC class 1 allele, as predicted by the prediction algorithm. WT1 peptide 122 comprises within it a known CD8+ epitope (126-134). In one embodiment, therefore, a modified peptide of the peptide that spans the WT1 amino acid residues 126-134 and contains a modified amino acid at positions may be used. Peptides used for alanine mutagenesis of WT1A (otherwise designated RFM) were named based on the position where the substitution was made. Examples of WT1 peptides which may be used are shown in Table 10 along with irrelevant peptides, RHAMM-R3 and EW.

TABLE-US-00010 TABLE 10 WT1A(RMF) RMFPNAPYL SEQ ID NO.: 1 WT1A1-B AMFPNAPYL SEQ ID NO.: 110 WT1A-3 RMAPNAPYL SEQ ID NO.: 111 WT1A-4 RMFANAPYL SEQ ID NO.: 112 WT1A-5 RMFPAAPYL SEQ ID NO.: 113 WT1A-7 RMFPNAAYL SEQ ID NO.: 114 WT1A-8 RMFPNAPAL SEQ ID NO.: 115 RHAMM-R3 ILSLELMKL SEQ ID NO.: 116 EW QLQNPSYDK SEQ ID NO.: 117 RSDELVRHHNMHQRNMTKL SEQ ID NO.: 118 PGCNKRYFKLSHLQMHSRKHTG SEQ ID NO.: 119 SGQARMFPNAPYLPSCLES SEQ ID NO.: 120 SGQAYMFPNAPYLPSCLES SEQ ID NO.: 121

[0137] Once a suitable peptide has been identified, the target antigen to be used for phage display library screening, that is, a peptide/HLA complex (for example, WT1 peptide/HLA-A0201) is prepared by bringing the peptide and the histocompatibility antigen together in solution to form the complex.

Selecting a High Affinity ScFV Against a WT1 Peptide

[0138] The next step is the selection of phage that bind to the target antigen of interest with high affinity, from phage in a human phage display library that either do not bind or that bind with lower affinity. This is accomplished by iterative binding of phage to the antigen, which is bound to a solid support, for example, beads or mammalian cells followed by removal of non-bound phage and by elution of specifically bound phage. In one embodiment, antigens are first biotinylated for immobilization to, for example, streptavidin-conjugated Dynabeads M-280. The phage library is incubated with the cells, beads or other solid support and non binding phage is removed by washing. Clones that bind are selected and tested.

[0139] Once selected, positive scFv clones are tested for their binding to HLA-A2/peptide complexes on live T2 cell surfaces by indirect flow cytometry. Briefly, phage clones are incubated with T2 cells that have been pulsed with Wt1-A peptide, or an irrelevant peptide (control). The cells are washed and then with a mouse anti-M13 coat protein mAb. Cells are washed again and labeled with a FITC-goat (Fab).sub.2 anti-mouse Ig prior to flow cytometry.

[0140] In other embodiments, the anti-WT1/A antibodies may comprise one or more framework region amino acid substitutions designed to improve protein stability, antibody binding, expression levels or to introduce a site for conjugation of therapeutic agents. These scFv are then used to produce recombinant human monoclonal Igs in accordance with methods known to those of skill in the art.

[0141] Methods for reducing the proliferation of leukemia cells is also included, comprising contacting leukemia cells with a WT1 antibody of the invention. In a related aspect, the antibodies of the invention can be used for the prevention or treatment of leukemia. Administration of therapeutic antibodies is known in the art.

Antibody Conjugates with Anti-Cancer Agents

[0142] Monoclonal antibodies represent the preferred vehicle for the targeted delivery of bioactive agents to cancer sites, including antibody-based delivery of cytotoxics, radionuclides or immunomodulatory cytokines. Conjugates of the antibodies of the invention with therapeutic agents, including without limitation, drugs (such as calecheamicin, aureastatin, doxorubicin), or toxins (such as ricin, diphtheria, gelonin) or radioisotopes emitting alpha or beta particles (such as, .sup.90I, .sup.131I, .sup.225Ac, .sup.213Bi, .sup.223Ra and .sup.227Th), inflammatory peptides (such as IL2, TNF, IFN-.gamma.) are encompassed by the invention.

[0143] Pharmaceutical Compositions and Methods of Treatment

[0144] WT1 antibodies of the present invention can be administered for therapeutic treatments to a patient suffering from a tumor or WT1-associated pathologic condition in an amount sufficient to prevent, inhibit, or reduce the progression of the tumor or pathologic condition. Progression includes, e.g, the growth, invasiveness, metastases and/or recurrence of the tumor or pathologic condition. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system. Dosing schedules will also vary with the disease state and status of the patient, and will typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours), or as indicated by the treating physician and the patient's condition.

[0145] The identification of medical conditions treatable by WT1 antibodies of the present invention is well within the ability and knowledge of one skilled in the art. For example, human individuals who are either suffering from a clinically significant leukemic disease or who are at risk of developing clinically significant symptoms are suitable for administration of the present WT1 antibodies. A clinician skilled in the art can readily determine, for example, by the use of clinical tests, physical examination and medical/family history, if an individual is a candidate for such treatment.

[0146] Non-limiting examples of pathological conditions characterized by WT1 expression include chronic myelocytic leukemia, acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Additionally, solid tumors, in general and in particular, tumors associated with mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma are amenable to treatment using WT1 antibodies.

[0147] In another embodiment, therefore, the present invention provides a method of treating a medical condition by administering a WT1 antibody of the present invention in combination with one or more other agents. For example, an embodiment of the present invention provides a method of treating a medical condition by administering a WT1 antibody of the present invention with an antineoplastic or antiangiogenie agent. The WT1 antibody can be chemically or biosynthetically linked to one or more of the antineoplastic or antiangiogenic agents.

[0148] Any suitable method or route can be used to administer a WT1 antibody of the present invention, and optionally, to coadminister antineoplastic agents and/or antagonists of other receptors. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration.

[0149] It is noted that a WT1 antibody of the present invention can be administered as a conjugate, which binds specifically to the receptor and delivers a toxic, lethal payload following ligand-toxin internalization.

[0150] It is understood that WT1 antibodies of the invention will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection may, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.

[0151] Other aspects of the invention include without limitation, the use of antibodies and nucleic acids that encode them for treatment of WT1 associated disease, for diagnostic and prognostic applications as well as use as research tools for the detection of WT1 in cells and tissues. Pharmaceutical compositions comprising the disclosed antibodies and nucleic acids are encompassed by the invention. Vectors comprising the nucleic acids of the invention for antibody-based treatment by vectored immunotherapy are also contemplated by the present invention. Vectors include expression vectors which enable the expression and secretion of antibodies, as well as vectors which are directed to cell surface expression of the antigen binding proteins, such as chimeric antigen receptors.

[0152] Cells comprising the nucleic acids, for example cells that have been transfected with the vectors of the invention are also encompassed by the disclosure.

[0153] For use in diagnostic and research applications, kits are also provided that contain a WT1 antibody or nucleic acids of the invention, assay reagents, buffers, and the like.

[0154] The method of the present invention will now be described in more detail with respect to representative embodiments.

Materials

[0155] Cell samples, cell lines and antibodies. After informed consent on Memorial Sloan-Kettering Cancer Center Institutional Review Board approved protocols, peripheral blood mononuclear cells (PBMC) from HLA-typed healthy donors and patients were obtained by Ficoll density centrifugation. The sources for obtaining human mesothelioma cell lines are described previously (29). The cell lines include: H-Meso1A, JMN, VAMT, H2452, H2373, H28, MSTO, Meso 11, Meso 34, Meso 37, Meso 47, and Meso 56. All cells were HLA typed by the Department of Cellular Immunology at Memorial Sloan-Kettering Cancer Center. Leukemia cell lines LAMA81, BV173, and 697, (WT1+, A0201+) were kindly provided by Dr. H. J. Stauss (University College London, London, United Kingdom). Melanoma cell line MeWo (WT1-, A201+), SKLY16 (B-cell lymphoma; WT1-, A0201+); K562, RwLeu4, and HL60, all WT1+ leukemias, and a TAP-deficient T2 cell line were obtained from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 supplemented with 5% FCS, penicillin, streptomycin, 2 mmol/L glutamine, and 2-mercaptoethanol at 37 C/5% CO2.

[0156] Monoclonal Ab against human HLA-A2 (clone BB7.2) conjugated to FITC or APC, and its isotype control mouse IgG2b/FITC or APC, to human or mouse CD3, CD19, CD56, CD33, CD34 (BD Biosciences, San Diego), goat F(ab)2 anti-human IgG conjugated with PE or FITC and goat F(ab)2 anti-mouse Ig's conjugated to fluorescent (In Vitrogen, City) were purchased. Mouse mAb to HLA-class I (W6/32) was obtained from the MSKCC Monoclonal antibody Core Facility.

[0157] Peptides. All peptides were purchased and synthesized by Genemed Synthesis, Inc. (San Antonio, Tex.). Peptides were >90% pure. (Table 1.) The peptides were dissolved in DMSO and diluted in saline at 5 mg/mL and frozen at -180 C. Biotinylated single chain WT1 peptide/HLA-A0201 and RHAMM-3/HLA-A0201 complexes were synthesized by refolding the peptides with recombinant HLA-A2 and beta2 microglobulen (.beta.2M) at the Tetramer facility at MSKCC.

[0158] Animals. Eight to 10 week-old NOD.Cg-Prkdc scid IL2rgtm1Wjl/SzJ mice, known as NOD scid gamma (NSG), were purchased from the Jackson Laboratory (Bar Harbor, Me.) or obtained from MSKCC animal breeding facility.

Methods

[0159] Flow cytometry analysis. For cell surface staining, cells were incubated with appropriate mAbs for 30 minutes on ice, washed, and incubated with secondary antibody reagents when necessary. Flow cytometry data were collected on a FACS Calibur (Becton Dickinson) and analyzed with FlowJo V8.7.1 and 9.4.8 software.

[0160] Selection and characterization of scFv specific for WT1 peptide/HLA-A0201 complexes. A human scFv antibody phage display library was used for the selection of mAb clones. In order to reduce the conformational change of MHC1 complex introduces by immobilizing onto plastic surfaces, a solution panning method was used in place of conventional plate panning. In brief, biotinylated antigens were first mixed with the human scFv phage library, then the antigen-scFv antibody complexes were pulled down by streptavidin-conjugated Dynabeads M-280 through a magnetic rack. Bound clones were then eluted and were used to infect E. coli XL1-Blue. The scFv phage clones expressed in the bacteria were purified (35, 36). Panning was performed for 3-4 cycles to enrich scFv phage clones binding to HLA-A0201/WT1 complex specifically. Positive clones were determined by standard ELISA method against biotinylated single chain HLA-A0201/WT1 peptide complexes. Positive clones were further tested for their binding to HLA-A2/peptide complexes on live cell surfaces by flow cytometry, using a TAP-deficient, HLA-A0201+ cell line, T2. T2 cells were pulsed with peptides (50 ug/ml) in the serum-free RPM11640 medium, in the presence of 20 .mu.g/ml .beta.2 M ON. The cells were washed, and the staining was performed in following steps.

[0161] The cells were first stained with purified scFv phage clones, and followed by staining with a mouse anti-M13 mAb, and finally the goat F(ab)2 anti-mouse Ig's conjugate to FITC. Each step of the staining was done between 30-60 minutes on ice and the cells were washed twice between each step of the staining.

[0162] Engineering full length mAb using the selected ScFv fragments. Full-length human IgG1 of the selected phage clones were produced in HEK293 and Chinese hamster ovary (CHO) cell lines, as described (37). In brief, antibody variable regions were subcloned into mammalian expression vectors, with matching human lambda or kappa light chain constant region and human IgG1 constant region sequences. Molecular weight of the purified full length IgG antibodies were measured under both reducing and non-reducing conditions by electrophoresis.

[0163] Engineering chimeric antigen receptors and immune effector cells. Nucleic acids that encode antibodies and antigen binding proteins identified herein can be used engineer recombinant immune effector cells. Methods and vectors to generate genetically modified T-cells, for example, are known in the art (See Brentjens et al., Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias in Blood 118(18):4817-4828, November 2011).

[0164] Characterization of the full-length human IgG1 for the WT1p/A2 complex. Initially, specificities of the fully human IgG1 mAbs for the WT1 peptide/A2 complex were determined by staining T2 cells pulsed with or without RMF or RHAMM-R3 control peptides, followed by secondary goat F(ab)2 anti-human IgG mAb conjugate to PE or FITC. The fluorescence intensity was measured by flow cytometry. The same method was used to determine the binding of the mAbs to fresh tumor cells and cell lines.

[0165] Radioimmunoassays. WT1 ab1 was labeled with 125-I (PerkinElmer) using the chloramine-T method (38). 100 .mu.g antibody was reacted with 1 mCi 125-I and 20 .mu.g chloramine-T, quenched with 200 .mu.g Na metabisulfite, then separated from free 125-I using a 10DG column (company) equilibrated with 2% bovine serum albumin in PBS. Specific activities of products were in the range of 7-8 mCi/mg.

[0166] Hematopoietic cell lines, adherent cell lines (harvested with a non-enzymatic cell stripper (name)), PBMCs from normal donors and AML patients were obtained as described. Cells were washed once with PBS and re-suspended in 2% human serum in PBS at 10.sup.7 cells/mL at 0.degree.. Cells (10.sup.6 tube in duplicate) were incubated with 125-I-labeled WT1 AB1 (1 .mu.g/mL) for 45 minutes on ice, then washed extensively with 1% bovine serum albumin in PBS at 0.degree.. To determine specific binding, a duplicate set of cells was assayed after pre-incubation in the presence of 50-fold excess unlabeled WT1 AB1 for 20 minutes on ice. Bound radioactivity was measured by a gamma counter, specific binding was determined, and the number of bound antibodies per cell was calculated from specific activity.

[0167] Antibody-dependent cellular cytotoxicity (ADCC). Target cells used for ADCC were T2 cells pulsed with or without WT1 or RHAAM-3 peptides, and tumor cell lines without peptide pulsing. WT1 ab1 or its isotype control human IgG1 at various concentrations were incubated with target cells and fresh PBMCs at different effector: target (E:T) ratio for 16 hrs. The supernatant were harvested and the cytotoxicity was measured by LDH release assay using Cytotox 96 non-radioreactive kit from Promega following their instruction. Cytotoxicity is also measured by standard 4 hours 51Cr-release assay.

[0168] Transduction and selection of luciferase/GFP positive cells. BV173 and JMN cells were engineered to express high level of GFP-luciferase fusion protein, using lentiviral vectors containing a plasmid encoding the luc/GFP (39). Using single cell cloning, only the cells showing high level GFP expression were selected by flow cytometry analysis and were maintained and used for the animal study.

[0169] Therapeutic trials of the WT1 ab1 in a human leukemia xenograft NSG model. Two million BV173 human leukemia cells were injected IV into NSG mice. On day 5, tumor engraftment was confirmed by firefly luciferase imaging in all mice that were to be treated; mice were then randomly divided into different treatment groups. On day 6 and day 10, mAb WT1 ab1 or the isotype control mAb were injected IV. In animals that also received human effector cells with or without mAb, cells (CD34 and CD3-depleted healthy donor human PBMCs) were injected IV into mice (10 cells/mouse) 4 hr before the mAb injections. Tumor growth was assessed by luminescence imaging once to twice a week, and clinical activity was assessed daily.

[0170] Selection and characterization of scFv specific for WT peptide/HLA-A0201 complexes. Selection of an WT1-specific scFV is achieved using a 9-mer T1-derived peptide comprising amino acids 126-134 (RMFPNAPYL, SEQ ID NO: 1) of WT1. This peptide has been shown to be processed and presented by HLA-A0201 to induce cytotoxic CD8.sup.+ T cells that are capable of killing WT1-positive tumor cells.

[0171] Representative data from a patient with AML after 6 vaccinations with a WT1 RMF peptide are shown in FIG. 2 as evidence that the WT1 peptides are immunogenic in humans. CD3 T cells were stimulated with WT1-A peptide (amino acids 126-134) and cytotoxicity was measured using a standard Cr release assay against 697 (A0201A WT1+) or SKLY-16 (A0201+WT1-) cell lines. The SKLY-16 cells pulsed with WT1-A or irrelevant peptide EW were used as positive and negative controls for the specificity of the killing. Effector: Target (E:T) ratios are indicated on the X-axis. Data demonstrates that T cells killed WT1+ tumor cells in a HLA-A0201-restricted manner.

[0172] Well established phage display libraries and screening methods known to those of skill in the art were used to select scFv fragments highly specific for an WT1-A peptide/HLA-A2 complex. In one embodiment, a human scFv antibody phage display library (7.times.10.sup.10 clones) was used for the selection of mAb clones. In order to reduce the conformational change of MHC1 complex introduced by immobilizing onto plastic surfaces, a solution panning method was used in place of conventional plate panning. In brief, biotinylated antigens were first mixed with the human scFv phage library, then the antigen-scFv phage antibody complexes were pulled down by streptavidin-conjugated Dynabeads M-280 through a magnetic rack.

[0173] Bound clones were then eluted and were used to infect E. coli XL1-Blue. The scFv phage clones expressed in the bacteria were purified (35, 36). Panning was performed for 3-4 cycles to enrich scFv phage clones binding to HLA-A0201/WT1 complex specifically. Positive clones were determined by standard ELISA method against biotinylated single chain HLA-A0201/WT1 peptide complexes (FIG. 3). Positive clones were further tested for their binding to HLA-A2/peptide complexes on live cell surfaces by flow cytometry, using a TAP-deficient, HLA-A0201.sup.+ cell line, T2. T2 cells were pulsed with peptides (50 .mu.g/ml) in serum-free RPM11640 medium, in the presence of 20 .mu.g/m .beta.2 M overnight. The cells were washed, and staining was performed as follows.

[0174] The cells were first stained with purified scFv phage clones, followed by staining with a mouse anti-M13 mAb, and finally, a goat F(ab)2 anti-mouse Ig conjugated to FITC. Each step of the staining was done for 30-60 minutes on ice. The cells were washed twice between each staining step. Results are shown in FIG. 4. The phage clone of WT1 ab1 was shown to bind to T2 cells pulsed with only WT1-A peptide (RMFPNAPYL: abbreviated hereinafter as RMF), but not to T2 cells alone, T2 cells pulsed with control EW peptide, or heteroclitic peptide WT1-A.

[0175] Binding affinity of the full-length IgG1 of WT1 ab1 to the peptide/A0201 complex was tested by titration of WT1 ab1 at indicated concentrations. T2 cells were pulsed with 50 .mu.g/ml or 10 .mu.g/ml, followed by secondary goat F(ab) anti-human IgG/PE. Results are shown in FIG. 5.

[0176] FIG. 6 shows the density of peptide/HLA-A0201 complex recognized by WT1 ab. T2 cells were pulsed overnight (ON) with RMF (upper panel) or RHAMM-R3 (lower panel) peptides at indicated concentrations, and binding of WT1 ab1, WT1 ab3 and WT1 ab5 at a concentration of 1 .mu.g/ml was analyzed by flow cytometry.

TABLE-US-00011 TABLE 11 Summary of phage panning for WTI/A2 Number Solution of single ELISA Number Rounds of clone positive of Unique Phage libraries panning screened Rate Clones scFv-spleen A 4 72 41/96 13 scFv-spleen B 4 47 3/47 2 scFv-spleen C 3 58 0/58 0 scFv-PBMC A 4 68 34/68 10 scFv-PBMC B 3 90 19/90 7 Fab-spleen A 4 12 2/12 0 Fab-spleen B 4 36 0/36 0 Fab-spleen C 4 24 2/24 1 Fab-spleen C 3 72 38/72 5 Fab-spleen D 4 72 4/72 1 Fab-spleen D 4 72 4/72 3

[0177] The positive scFv clones were tested for their binding to HLA-A2/peptide complexes on live cell surfaces by indirect flow cytometry on: (i) a TAP deficient HLA-A0201.sup.+ T2 cells pulsed with WT1 peptide or irrelevant peptide; (ii) a WT1+ HLAA0201.sup.+ cell lines such as BV173, U266 and control WTI HLA-A0201.sup.+ cell line SKLY16, or WT1.sup.+ HLA-A0201 cell line, K562, without pulsing with the peptide. The latter determine the recognition and binding affinity of the scFv to the naturally processed WT1p/A2 complex on tumor cells.

[0178] A total of 28 phage clones were screened for their ability to produce mAb specific for the WTI-A peptide/A2 complex. The recognition of the WT1 p/A2 complex on live cells was measured by the binding of the phage scFv to T2 cells pulsed with the WTI-A peptide and the other HLA-A2-binding peptides (50 .mu.g/ml). These include: T2 cells alone; T2 cells pulsed with WTI-A peptide; T2 cells pulsed with heteroclitic peptide WT1-A1; T2 cells pulsed with irrelevant EW peptide (HLA-A0201-binding 9-mer peptide, derived from Ewing sarcoma) or RHAMM-R3 (FIG. 4).

TABLE-US-00012 TABLE 12 Positive for binding toT2 Selected for Construction Clone # pulsed with WT1A of full-length IgG1 1 + 2 + 3 + + 4 + 5 + + 6 + 7 8 + 9 10 11 12 13 + + 14 15 + + 16 17 + 18 + + 19 + 20 + 21 22 + 23 + + 24-28

Engineering Full Length mAb Using the Selected ScFv Fragments.

[0179] Phage display technology allows for the rapid selection and production of antigen-specific scFv and Fab fragments, which are useful in and of themselves, or which can be further developed to provide complete antibodies, antigen binding proteins or antigen binding fragments thereof. Complete mAbs with Fc domains have a number of advantages over the scFv and Fab antibodies. First, only full length Abs exert immunological function such as CDC and ADCC mediated via Fc domain. Second, bivalent mAbs offer stronger antigen-binding affinity than monomeric Fab Abs. Third, plasma half-life and renal clearance will be different with the Fab and bivalent mAb. The particular features and advantages of each can be matched to the planned effector strategy. Fourth, bivalent mAb may be internalized at different rates than scFv and Fab, altering immune function or carrier function. Alpha emitters, for example, do not need to be internalized to kill the targets, but many drugs and toxins will benefit from internalization of the immune complex. In one embodiment, therefore, once scFv clones specific for WT1p/A2 were obtained from phage display libraries, a full length IgG mAb using the scFv fragments was produced.

[0180] To produce recombinant human monoclonal IgG in Chinese hamster ovary (CHO) cells, a full length IgG mAb was engineered based on a method known to those of skill in the art (Tomomatsu et al., Production of human monoclonal antibodies against FceRIa by a method combining in vitro immunization with phage display. Biosci Biotechnol Biochem 73(7): 1465-1469 2009). Briefly, antibody variable regions were subcloned into mammalian expression vectors (FIG. 7), with matching Lambda or Kappalight chain constant sequences and IgG1 subclass Fc (for example, see Table 9) (33,34). Purified full length IgG antibodies showed expected molecular weight under both reducing and non-reducing conditions (FIG. 8). Kinetic binding analysis (35) confirmed specific binding of full length IgG to WT1/A2, with a K.sub.D in nanomolar range (FIGS. 9 and 10.)

Example 1

Selection of ScFv Specific for WT1p/A2 Complex Using a Fully Human Phage Display Library.

[0181] Phage display against HLA-A0201/WT1 peptide complex was performed for 3-4 panning rounds to enrich the scFv phage clones binding to HLA-A0201/WT1 peptide complex specifically. Individual scFv phage clones positive for the WT1 peptide/A2 complex were determined by ELISA and the clones that possessed unique DNA coding sequences were subjected to further characterization. To test if the ScFv bound to the WT1p/A2 complex on live cells, the positive phage clones were tested for binding to a TAP deficient, HLA-A0201-positive cell line, T2. T2 cells can only present the exogenous peptides and therefore have been widely used for detection of specific epitopes presented by HLA-A2 molecules. A total 35 phage clones were screened on T2 cells and 15 clones showed specific binding to T2 cells pulsed with only WT1 RMF peptide, but not to T2 cells alone or pulsed with control RHAMM-3 peptide (FIG. 4). The scFv phage clones were unable to bind to several tumor cell lines that are WT1- and HLA-A2 positive suggesting the affinity of the ScFv was weak, compared to full-length bivalent mAb.

Example 2

Generation of Full-Length Human IgG1.

[0182] Immunological function such as CDC and ADCC depend on the Fc domain of bivalent IgG. In addition, bivalent mAbs offer stronger antigen-binding avidity than monomeric scFv Abs. Therefore, 6 ScFv phage clones among 15 positive phage clones were selected to produce the full-length human monoclonal IgG1 in HEK293 and Chinese hamster ovary (CHO) cells. In brief, variable regions of the mAbs were subcloned into mammalian expression vectors with matching human lambda or kappa light chain constant region and human IgG1 constant region sequences. Purified full length IgG antibodies showed expected molecular weight under both reducing and non-reducing conditions (FIG. 8). Five clones were successfully engineered into human IgG1.

Example 3

Specificity and Binding Avidity of the IgG1 mAb

Binding to Human Cell Lines.

[0183] T2 cells, pulsed with or without RMF or RHAMM-3 peptides initially were used to determine the binding specificity of the mAb. Three out of five human IgG1, including WT1 ab1, showed specific binding to the T2 cells that were pulsed only with WT1 peptide, but not to T2 alone or T2 pulsed with control peptide RHAMM-R3. The binding avidity of the mAb were substantially enhanced (50 to 100 fold), compared to their parental scFv phage clones. Two mAbs among the five showed binding to T2 cells alone or pulsed with the control peptide RHAMM-R3, although the binding was greatly enhanced by pulsing the cells with RMF peptide. This suggested that these two mAb also had high avidity for epitopes on the HLA-A2 molecule alone and therefore were excluded from further investigation. This was not unexpected, as it has been a common problem for producing such mAb against peptide/MHC complexes, given the predominance of the MHC class I molecule epitopes within the complexes. It also suggests that the precise specificity of the mAb for the complexes might not be determined easily at the scFv stage, due to the lower affinity compared to the bivalent IgG1 mAb.

[0184] The binding affinity of the three remaining mAb specific for the WT1p/A2 complex first was investigated on T2 cells pulsed with or without RMF and control RHAMM-R3 peptides (50 ug/ml) by titration of the mAbs. Mab WT1 ab1 showed the strongest binding, down to a concentration of 0.01 ug/ml. Isotype control human IgG1 showed no binding at any concentrations tested (FIG. 5). In addition to WT1 ab1, the two other mAb, WT1 ab3 and WT1 ab5, showed specific binding at a range of <1 ug/ml of the mAb concentrations used. The specific recognition of the mAb also depended on the antigenic density on the cell surface. T2 cells were pulsed with RMF or R3 peptides at 50, 25, 12.5, 6.25, 3.13 and 1.6 ug/ml; the test mAb were used at 1 ug/ml for the T2 binding assay. WT1 ab1 could detect the RMF peptide/A2 complex on T2 cells in a concentration-dependent manner at concentrations as low as 1.6 ug/ml, with significantly higher fluorescence intensity than the other 2 mAb (FIG. 6). These results further confirmed that the WT1 ab1 possessed the highest avidity for the RMFp/A0201 complex.

Example 4

Epitope Mapping.

[0185] To investigate with more precision the epitope for WT1 ab1 recognition, RMF peptides were substituted at positions 1, 3, 4, 5, 6, 7 and 8 with alanine and pulsed onto T2 cells and were tested for binding of WT1 ab1. Positions 2 and 9 of the RMF were left intact, as these are the anchor residues for peptide binding to the HLA-A0201 molecule. Except for position 1, alanine substitutions at other positions did not greatly affect the binding of the WT1 ab1, as compared to the native RMF peptide (FIG. 19). However, substitution of position 1 by either alanine (WT1-A1-B) or tyrosine (WT1-A1), completely abrogated the binding of WT1 ab1. The loss of binding was not due to the reduction of peptide binding affinity to the HLA-A2 molecule, as both peptides showed the strongest binding in T2 stabilization assay using the mAb specific for the HLA-A2 molecule, clone BB7 (FIG. 20). These results show that the arginine at position 1 of the RMF peptide is one of the most crucial for the WT1 ab1 recognition. The role of the residues at positions #2 and 9, could not be assessed.

[0186] The next important question was whether WT1 ab1 was able to recognize naturally processed WT1 epitope RMF presented by HLA-A0201 molecules on the cell surface. A panel of cell lines was selected based on the expression of WT1 mRNA and HLA genotyping (Table 12).

TABLE-US-00013 TABLE 12 Ratio of HLA-A2 WT1 WT1 AB BB7.2: genotype mRNA binding Isotype Mesothelioma/solid tumor JMN + + + 248 Meso 37 + + + 68 Meso 47 + (02xx) + + 17 H2452 + + + 20 Meso34 + + + 37.3 Meso-56 + (02xx) + + 23 H2373 + + - 1.6 MSTO - + - 1.4 VAMT - 3+ - NT Mewo + - - 3 Leukemias and other hematopoietic cell lines BV173 + ++ + 196 BA25 + ? + 117.5 ALL-3 + + + 60 U266 + + - 1.8 697 + 5+ - 4.1 LAMA + 2+ - 6 SKLY-16 + - - 1.9 HL-60 - 3+ - 0.4 K562 - 2+ - 1.5 T2 + NT - >20

[0187] WT1 mRNA expression level was estimated according to a previous study (Rena), by quantitative RT-PCR.

[0188] Among 7 human mesothelioma cell lines that are positive for both HLA-A0201 and WT1 mRNA, WT1 ab1 bound to 6 out of 7 cell lines, but not to the cells that were either HLA-A0201 negative (MSTO and VAMT) or WT1 mRNA negative, such as melanoma cell line, Mewo (FIG. 21).

[0189] Similarly, among 9 leukemia cell lines tested, WT1 ab1 bound to 3 cell lines BV173 (FIG. 22), BA25 and ALL-3, that are positive for both WT1 mRNA and HLA-A0201, but not to HLA-A2-negative cell lines HL60 and K562, that have been demonstrated to express a high level of WT1 transcripts in numerous studies.

[0190] As expected, intensity of binding of the WT1 AB1 also appeared to be directly associated with the expression level of HLA-A0201 molecule, as shown in mesothelioma cells H2373, leukemia cell lines 697 and LAMA, and myeloma cell line U266. Although these cell lines were positive for both WT1 transcripts and HLA-A2, the expression level of the HLA-A2 was low (Table 12) and the mAb did not show binding. On the other hand, the results obtained with T2 cells argue against the possibility of WT1 ab1 binding to HLA-A0201 alone as T2 cells expressed a high level of HLA-A2 molecule. Notably, WT1 ab1 did not bind to T2 cells alone or pulsed with R3 and other HLA-A0201-binding peptides such as Ewing sarcoma-derived (EW) or the heteroclitic peptide for the RMF peptide, WT1-A1; these two peptides have been shown to have higher affinity for the HLA-A0201 molecule in T2 stabilization assay (28). These results provided strong evidences that WT1 ab1 recognition was specific for epitopes jointly composed of the RMF peptide and the A0201 molecule in a complex. The binding of the other two mAb, WT1 ab3 and WT1 ab5, to the BV173 and JMN cells was also weaker than WT1 ab1.

Example 5

Quantitation of WT1 ab1 Binding Sites on Cells.

[0191] A radioimmunoassay using 1251-labeled WT1 ab1 was used to confirm the specificity of the antibody for WT1+ HLA-A0201+ cell lines, to determine an affinity constant and to assess the number of antibody binding sites per cell on a panel of cell lines. Scatchard analysis based on binding to JMN cells showed an avidity constant of about 0.2 nM (FIG. 23). This number was confirmed by interferometry using a Forte Bio device. 125-I-labeled WT1 ab1 was used to confirm the specificity of the antibody for WT1+ HLA-A0201+ cell lines, and to assess the number of antibody binding sites on a panel of cell lines (FIG. 24). Because we cannot determine whether the bivalent mAb is binding to 1 or 2 complexes on the surface, total epitopes per cell could be as high as twice the number of mAb binding sites. Again, WT1 ab1 bound to JMN, ALL-3, BA25, BV173, which are positive for both HLA-A0201 and WT1 mRNA, but not HLA-A0201 negative (HL60) or WT1 mRNA negative (SKLY-16) cells. WT1 ab1 did not bind to 697 cells, which are both HLA-A0201 and WT1 positive, but contain low levels of HLA-A0201 (Table 12), confirming that a certain level of total MHC complex is needed to present sufficient WT1 peptide for WT1 ab1 binding. T2 pulsed with RMF bound the highest number of mAb (50,000 per cell), followed by JMN cells which bound 6.times.103 WT1 ab1 molecules per cell, translating to between 6.times.103 and 1.2.times.104 RMF peptide/A2 complexes per cell assuming monovalent or bivalent antibody binding, respectively. The three positive leukemia cell lines bound between 1.times.103 and 2.times.103 WT1 ab1 molecules, or 2.times.103-4.times.103 binding sites (FIG. 24). These results were confirmed by quantitative flow cytometry.

Example 6

WT1 Ab1 Binding to Leukemic Patient Samples.

[0192] We next investigated if WT1 ab1 is able to detect the RMF epitope on primary AML cells. Radioimmunoassay showed significant binding of the WT1 AB1 to AML blasts of patient 1, who is HLA-A2 positive and WT1 mRNA.sup.+. WT1 ab1 bound to CD33+ and CD34+ double positive cells that account for more than 83% of the whole cell populations (FIG. 25). WT1 ab1 did not bind to the cells of 3 other patients who are either HLA-A2 positive but mRNA negative or HLA-A2 negative. WT1 ab1 did not bind to PBMCs from either HLA-A2 positive or negative healthy donors. The results were confirmed by flow cytometry analysis. WT1 AB1 did not show significant binding to the blasts from the patients who were A0201 negative (FIG. 26). The results were consistent with the results obtained with mRNA expression of the cells. These data confirm that the level of RMFp/HLA-A0201 on the surface of leukemia cells is adequate to allow reactivity with the WT1 ab1 and the levels on WT1 negative healthy cells is not significant.

Example 7

WT1 AB1 Mediates ADCC Against Tumor Cells

[0193] ADCC is considered to be one of the major effector mechanisms of therapeutic mAb in humans. In the presence of human PBMC, WT1 ab1 mediated dose-dependent PBMC ADCC against the T2 cells loaded with RMF peptide, but not T2 cells alone or T2 cells pulsed with control R3 peptide (FIG. 27). Importantly, WT1 ab1 was able to mediate ADCC against naturally presented RMF epitope by HLA-A0201 molecule on tumor cells, such as the mesothelioma cell line, JMN (FIG. 33), and the leukemia cell line BV173 (FIG. 34), but not the HLA-A2 negative cells MSTO (FIG. 28) or HL-60 (FIG. 29). The killing was consistently observed at 1 .mu.g/ml or below of WT1 ab1 using PBMCs as effector cells from multiple healthy donors. Importantly, WT1 ab1 also killed primary A0201-positive AML blasts that were positive for the WT1 ab1 binding, but not the blasts that were HLA-A0201 negative (FIG. 30). These results demonstrated that WT1 ab1 mediates specific ADCC against cells that naturally express RMF and HLA-A0201 complex at physiologic levels as well as on cell lines.

Example 8

WT1 AB1 Eliminates Human Leukemia Cells in NSG Mice

[0194] The efficacy of WT1 ab1 in vivo was tested in NOD SCID gamma (NSG) mice xenografted intravenously 6 days previously with BV173 bcr/abl positive acute lymphoblastic leukemia. At the time of treatment, mice had leukemia in their liver, spleen, and BM visible by luciferase imaging. NSG mice lack mature B-, T- and NK-cells, and we hypothesized that introducing human effector cells (CD3-, CD34-, PBMCs) along with WT1 ab1 treatment would recapitulate in vivo the ADCC-mediated anti-tumor effects observed in vitro. Injection of effectors along with two 100 .mu.g doses of WT1 ab1 nearly ablated tumor growth compared to controls (FIG. 31). This effect was durable over the course of the experiment (FIG. 32). Interestingly, early on in the trials, effector cells alone or combined with control IgG appeared to promote more rapid growth of leukemia relative to mice injected with leukemia alone, demonstrating that the anti-tumor effect was unrelated to the effectors by themselves. Several of the mice given effectors (with or without control mAb) died early in the experiment with massive infiltration of the BV173.

[0195] Surprisingly, WT1 ab1 treatment without human effectors also dramatically reduced tumor burden as well as the WT1 ab1 combined with effectors for approximately 30 days (FIG. 32), though tumors eventually relapsed far more quickly in the WT1 ab1 alone group, when compared to WT1 ab1 combined with effectors group (FIG. 32). We confirmed the effect of WT1 ab1 alone and titrated the dosage to evaluate potency. WT1 ab1 alone produced a marked reduction in tumor burden at early time points at all doses tested (25-100 .mu.g times 2 doses). Tumors in all treatment groups relapsed slowly after antibody therapy was stopped; and by day 23 (13 days after the last antibody injection), significantly more tumor relapse could be observed in the 25 .mu.g group compared to the 100 .mu.g dose group, indicating a dose-response to WT1 ab1 therapy (FIG. 33). Before treatment, mice displayed the largest tumor burden in the liver, which was quickly cleared by WT1 ab1. Upon relapse, tumor in the highest dose group appeared to develop mainly in bone marrow, while tumor returned more quickly to the liver in mice treated with the lowest dose.

Example 9

[0196] Engineering Antibodies to Enhance their Cytotoxic Abilities.

[0197] Bispecific antibodies are constructed that recognize both WT1/A2 complex and CD3 on immune T cells as described (43,44) with a human IgG1 Fc. Bispecific antibodies are expected to recruit and target cytotoxic T cells to WT1/A2 positive cancer cells, while maintaining Fc effector functions and long half life in vivo. Three mechanisms are involved in the specific killing of cancer cells mediated by bispecific antibodies: i) killing by activated T cells; ii) ADCC activity; iii) CDC activity. Other formats of bispecific antibodies can be constructed, such tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb), and fusion protein with human serum albumin (45, 46, 47, 48), but are devoid of Fc effector functions with distinct pharmacokinetic profiles.

[0198] WT1/A2 target specific-ADCC activity is enhanced by expressing antibodies recombinantly in glycol-engineered CHO cells as described in U.S. Pat. Nos. 8,025,879; 8,080,415; and 8,084,022. The modified oligosaccharide N-glycan on Asn297 alters effector functions as follows: 1) higher affinity binding to CD16/FcRIIIa for improved ADCC activity mediated by human Natural Killer cells; 2) reduced binding affinity to CD32b/FcRIIb, an inhibitory receptor expressed in multiple types of immune cells (except NK cells), for improved ADCC activity mediated by effector cells such as neutrophils and antigen presentation by macrophage and DC cells (50, 51, 52). Enhanced antibodies are expected to achieve better efficacy for cancer treatment in vivo.

[0199] Glycosylation (specifically fucosylation) variants of IgG Fc can be produced using host expression cells and methods described in U.S. Pat. Nos. 8,025,879; 8,080,415; and 8,084,022, the contents of which are incorporated by reference. Briefly, messenger RNA (mRNA) coding for heavy or light chain of the antibodies disclosed herein, is obtained by employing standard techniques of RNA isolation purification and optionally size based isolation. cDNAs corresponding to mRNAs coding for heavy or light chain are then produced and isolated using techniques known in the art, such as cDNA library construction, phage library construction and screening or RT-PCR using specific relevant primers. In some embodiments, the cDNA sequence may be one that is wholly or partially manufactured using known in vitro DNA manipulation techniques to produce a specific desired cDNA. The cDNA sequence can then be positioned in a vector which contains a promoter in reading frame with the gene and compatible with the low fucose-modified host cell.

[0200] Numerous plasmids that contain appropriate promoters, control sequences, ribosome binding sites, and transcription termination sites, and optionally convenient markers are known in the art, these include but are not limited to, vectors described in U.S. Pat. Nos. 4,663,283 and 4,456,748. In one embodiment, the cDNA coding for the light chain and that coding for the heavy chain may be inserted into separate expression plasmids. In an alternative embodiment, the cDNA coding for the light chain and that coding for the heavy chain may be inserted together in the same plasmid, so long as each is under suitable promoter and translation control. Results are shown in FIG. 34.

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Sequence CWU 1

1

23819PRTHomo sapiens 1Arg Met Phe Pro Asn Ala Pro Tyr Leu1 5210PRTHomo sapiens 2Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser1 5 10317PRTHomo sapiens 3Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly410PRTHomo sapiens 4Arg Ile Pro Pro Tyr Tyr Gly Met Asp Val1 5 10530DNAHomo sapiens 5ggaggcacct tcagcagcta tgctatcagc 30651DNAHomo sapiens 6gggatcatcc ctatctttgg tacagcaaac tacgcacaga agttccaggg c 51730DNAHomo sapiens 7cggattcccc cgtactacgg tatggacgtc 30813PRTHomo sapiens 8Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr1 5 1097PRTHomo sapiens 9Arg Ser Asn Gln Arg Pro Ser1 51011PRTHomo sapiens 10Ala Ala Trp Asp Asp Ser Leu Asn Gly Val Val1 5 101139DNAHomo sapiens 11tctggaagca gctccaacat cggaagtaat tatgtatac 391221DNAHomo sapiens 12aggagtaatc agcggccctc a 211333DNAHomo sapiens 13gcagcatggg atgacagcct gaatggtgtg gta 3314119PRTHomo sapiens 14Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Ile Pro Pro Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11515357DNAHomo sapiens 15caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagacggatt 300cccccgtact acggtatgga cgtctggggc caagggacca cggtcaccgt ctcctca 35716111PRTHomo sapiens 16Gln Thr Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Ser Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Pro Arg65 70 75 80Ser Val Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 11017333DNAHomo sapiens 17cagactgtgg tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccaacagctc 120ccaggaacgg cccccaaact cctcatctat aggagtaatc agcggccctc aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggccccgg 240tccgtggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtgtggta 300ttcggcggag ggaccaagct gaccgtccta ggt 33318250PRTHomo sapiens 18Gln Thr Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Arg Ser Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Pro Arg65 70 75 80Ser Val Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser 100 105 110Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu 115 120 125Glu Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 130 135 140Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe145 150 155 160Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 165 170 175Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala 180 185 190Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 195 200 205Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 210 215 220Tyr Tyr Cys Ala Arg Arg Ile Pro Pro Tyr Tyr Gly Met Asp Val Trp225 230 235 240Gly Gln Gly Thr Thr Val Thr Val Ser Ser 245 25019750DNAHomo sapiens 19cagactgtgg tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccaacagctc 120ccaggaacgg cccccaaact cctcatctat aggagtaatc agcggccctc aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggccccgg 240tccgtggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtgtggta 300ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360ggcggctctg gtggtggatc cctcgagatg gcccaggtgc agctggtgca gtctggggct 420gaggtgaaga agcctgggtc ctcggtgaag gtctcctgca aggcttctgg aggcaccttc 480agcagctatg ctatcagctg ggtgcgacag gcccctggac aagggcttga gtggatggga 540gggatcatcc ctatctttgg tacagcaaac tacgcacaga agttccaggg cagagtcacg 600attaccgcgg acgaatccac gagcacagcc tacatggagc tgagcagcct gagatctgag 660gacacggccg tgtattactg tgcgagacgg attcccccgt actacggtat ggacgtctgg 720ggccaaggga ccacggtcac cgtctcctca 7502012PRTHomo sapiens 20Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn1 5 102118PRTHomo sapiens 21Arg Thr Tyr Tyr Gly Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10 15Lys Ser229PRTHomo sapiens 22Gly Arg Leu Gly Asp Ala Phe Asp Ile1 52336DNAHomo sapiens 23ggggacagtg tctctagcaa cagtgctgct tggaac 362454DNAHomo sapiens 24aggacatact acgggtccaa gtggtataat gattatgcag tatctgtgaa aagt 542527DNAHomo sapiens 25ggtcgcttag gggatgcttt tgatatc 272611PRTHomo sapiens 26Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 10277PRTHomo sapiens 27Ala Ala Ser Ser Leu Gln Ser1 5289PRTHomo sapiens 28Gln Gln Ser Tyr Ser Thr Pro Leu Thr1 52933DNAHomo sapiens 29cgggcaagtc agagcattag cagctattta aat 333021DNAHomo sapiens 30gctgcatcca gtttgcaaag t 213127DNAHomo sapiens 31caacagagtt acagtacccc tctcact 2732121PRTHomo sapiens 32Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Gly Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Arg Gly Arg Leu Gly Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val Ser Ser 115 12033363DNAHomo sapiens 33caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacgggtc caagtggtat 180aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300agaggtcgct taggggatgc ttttgatatc tggggccaag ggacaatggt caccgtctct 360tca 36334108PRTHomo sapiens 34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 10535324DNAHomo sapiens 35gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta cccctctcac tttcggcgga 300gggaccaaag tggatatcaa acgt 32436250PRTHomo sapiens 36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg Ser Arg Gly Gly 100 105 110Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met 115 120 125Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser 130 135 140Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser145 150 155 160Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu 165 170 175Glu Trp Leu Gly Arg Thr Tyr Tyr Gly Ser Lys Trp Tyr Asn Asp Tyr 180 185 190Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys 195 200 205Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala 210 215 220Val Tyr Tyr Cys Ala Arg Gly Arg Leu Gly Asp Ala Phe Asp Ile Trp225 230 235 240Gly Gln Gly Thr Met Val Thr Val Ser Ser 245 25037750DNAHomo sapiens 37gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta cccctctcac tttcggcgga 300gggaccaaag tggatatcaa acgttctaga ggtggtggtg gtagcggcgg cggcggctct 360ggtggtggtg gatccctcga gatggcccag gtacagctgc agcagtcagg tccaggactg 420gtgaagccct cgcagaccct ctcactcacc tgtgccatct ccggggacag tgtctctagc 480aacagtgctg cttggaactg gatcaggcag tccccatcga gaggccttga gtggctggga 540aggacatact acgggtccaa gtggtataat gattatgcag tatctgtgaa aagtcgaata 600accatcaacc cagacacatc caagaaccag ttctccctgc agctgaactc tgtgactccc 660gaggacacgg ctgtgtatta ctgtgcaaga ggtcgcttag gggatgcttt tgatatctgg 720ggccaaggga caatggtcac cgtctcttca 7503810PRTHomo sapiens 38Gly Tyr Ser Phe Thr Asn Phe Trp Ile Ser1 5 103917PRTHomo sapiens 39Arg Val Asp Pro Gly Tyr Ser Tyr Ser Thr Tyr Ser Pro Ser Phe Gln1 5 10 15Gly4012PRTHomo sapiens 40Val Gln Tyr Ser Gly Tyr Tyr Asp Trp Phe Asp Pro1 5 104130DNAHomo sapiens 41ggatacagct tcaccaactt ctggatcagc 304251DNAHomo sapiens 42agggttgatc ctggctactc ttatagcacc tacagcccgt ccttccaagg c 514336DNAHomo sapiens 43gtacaatata gtggctacta tgactggttc gacccc 364413PRTHomo sapiens 44Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn1 5 10457PRTHomo sapiens 45Ser Asn Asn Gln Arg Pro Ser1 54611PRTHomo sapiens 46Ala Ala Trp Asp Asp Ser Leu Asn Gly Trp Val1 5 104739DNAHomo sapiens 47tctggaagca gctccaacat cggaagtaat actgtaaac 394821DNAHomo sapiens 48agtaataatc agcggccctc a 214933DNAHomo sapiens 49gcagcatggg atgacagcct gaatggttgg gtg 3350121PRTHomo sapiens 50Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Glu1 5 10 15Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Phe 20 25 30Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Val Asp Pro Gly Tyr Ser Tyr Ser Thr Tyr Ser Pro Ser Phe 50 55 60Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Val Gln Tyr Ser Gly Tyr Tyr Asp Trp Phe Asp Pro Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12051363DNAHomo sapiens 51cagatgcagc tggtgcagtc cggagcagag gtgaaagagc ccggggagtc tctgaggatc 60tcctgtaagg gttctggata cagcttcacc aacttctgga tcagctgggt gcgccagatg 120cccgggaaag gcctggagtg gatggggagg gttgatcctg gctactctta tagcacctac 180agcccgtcct tccaaggcca cgtcaccatc tcagctgaca agtctaccag cactgcctac 240ctgcagtgga acagcctgaa ggcctcggac accgccatgt attactgtgc gagagtacaa 300tatagtggct actatgactg gttcgacccc tggggccagg gaaccctggt caccgtctcc 360tca 36352111PRTHomo sapiens 52Gln Ala Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 11053333DNAHomo sapiens 53caggctgtgg tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga agtaatactg taaactggta ccagcaggtc 120ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttgggtg 300ttcggcggag ggaccaagct gaccgtccta ggt 33354253PRTHomo sapiens 54Gln Ala Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30Thr Val Asn Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser 100 105

110Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Leu Glu Met Ala Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys 130 135 140Glu Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser145 150 155 160Phe Thr Asn Phe Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly 165 170 175Leu Glu Trp Met Gly Arg Val Asp Pro Gly Tyr Ser Tyr Ser Thr Tyr 180 185 190Ser Pro Ser Phe Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Thr 195 200 205Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala 210 215 220Met Tyr Tyr Cys Ala Arg Val Gln Tyr Ser Gly Tyr Tyr Asp Trp Phe225 230 235 240Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 25055759DNAHomo sapiens 55caggctgtgg tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga agtaatactg taaactggta ccagcaggtc 120ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttgggtg 300ttcggcggag ggaccaagct gaccgtccta ggttctagag gtggtggtgg tagcggcggc 360ggcggctctg gtggtggtgg atccctcgag atggcccaga tgcagctggt gcagtccgga 420gcagaggtga aagagcccgg ggagtctctg aggatctcct gtaagggttc tggatacagc 480ttcaccaact tctggatcag ctgggtgcgc cagatgcccg ggaaaggcct ggagtggatg 540gggagggttg atcctggcta ctcttatagc acctacagcc cgtccttcca aggccacgtc 600accatctcag ctgacaagtc taccagcact gcctacctgc agtggaacag cctgaaggcc 660tcggacaccg ccatgtatta ctgtgcgaga gtacaatata gtggctacta tgactggttc 720gacccctggg gccagggaac cctggtcacc gtctcctca 7595610PRTHomo sapiens 56Gly Tyr Asn Phe Ser Asn Lys Trp Ile Gly1 5 105717PRTHomo sapiens 57Ile Ile Tyr Pro Gly Tyr Ser Asp Ile Thr Tyr Ser Pro Ser Phe Gln1 5 10 15Gly589PRTHomo sapiens 58His Thr Ala Leu Ala Gly Phe Asp Tyr1 55930DNAHomo sapiens 59ggctacaact ttagcaacaa gtggatcggc 306051DNAHomo sapiens 60atcatctatc ccggttactc ggacatcacc tacagcccgt ccttccaagg c 516127DNAHomo sapiens 61cacacagctt tggccggctt tgactac 276211PRTHomo sapiens 62Arg Ala Ser Gln Asn Ile Asn Lys Trp Leu Ala1 5 10637PRTHomo sapiens 63Lys Ala Ser Ser Leu Glu Ser1 5648PRTHomo sapiens 64Gln Gln Tyr Asn Ser Tyr Ala Thr1 56533DNAHomo sapiens 65cgggccagtc agaatatcaa taagtggctg gcc 336621DNAHomo sapiens 66aaggcgtcta gtttagaaag t 216724DNAHomo sapiens 67caacaatata atagttatgc gacg 2468118PRTHomo sapiens 68Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn Phe Ser Asn Lys 20 25 30Trp Ile Gly Trp Val Arg Gln Leu Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Ala Ile Ile Tyr Pro Gly Tyr Ser Asp Ile Thr Tyr Ser Pro Ser Phe 50 55 60Gln Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr Ala Tyr65 70 75 80Leu His Trp His Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Val Arg His Thr Ala Leu Ala Gly Phe Asp Tyr Trp Gly Leu Gly Thr 100 105 110Leu Val Thr Val Ser Ser 11569354DNAHomo sapiens 69caggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggagagtc tctgaagatc 60tcctgtaagg gttctggcta caactttagc aacaagtgga tcggctgggt gcgccaattg 120cccgggagag gcctggagtg gatagcaatc atctatcccg gttactcgga catcacctac 180agcccgtcct tccaaggccg cgtcaccatc tccgccgaca cgtccattaa caccgcctac 240ctgcactggc acagcctgaa ggcctcggac accgccatgt attattgtgt gcgacacaca 300gctttggccg gctttgacta ctggggcctg ggcaccctgg tcaccgtctc ctca 35470107PRTHomo sapiens 70Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn Lys Trp 20 25 30Leu Ala Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ala Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10571321DNAHomo sapiens 71gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcaca 60atcacttgcc gggccagtca gaatatcaat aagtggctgg cctggtatca gcagagacca 120gggaaagccc ctcagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatct 180aggttcagcg gcagtggatc tgggacagaa tacactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacaa tataatagtt atgcgacgtt cggccaaggg 300accaaggtgg aaatcaaacg t 32172246PRTHomo sapiens 72Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Asn Lys Trp 20 25 30Leu Ala Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Gln Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ala Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Arg Gly Gly Gly 100 105 110Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Met Ala 115 120 125Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 130 135 140Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn Phe Ser Asn Lys145 150 155 160Trp Ile Gly Trp Val Arg Gln Leu Pro Gly Arg Gly Leu Glu Trp Ile 165 170 175Ala Ile Ile Tyr Pro Gly Tyr Ser Asp Ile Thr Tyr Ser Pro Ser Phe 180 185 190Gln Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Ile Asn Thr Ala Tyr 195 200 205Leu His Trp His Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 210 215 220Val Arg His Thr Ala Leu Ala Gly Phe Asp Tyr Trp Gly Leu Gly Thr225 230 235 240Leu Val Thr Val Ser Ser 24573738DNAHomo sapiens 73gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcaca 60atcacttgcc gggccagtca gaatatcaat aagtggctgg cctggtatca gcagagacca 120gggaaagccc ctcagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatct 180aggttcagcg gcagtggatc tgggacagaa tacactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacaa tataatagtt atgcgacgtt cggccaaggg 300accaaggtgg aaatcaaacg ttctagaggt ggtggtggta gcggcggcgg cggctctggt 360ggtggtggat ccctcgagat ggcccaggtg cagctggtgc agtctggagc agaggtgaaa 420aagcccggag agtctctgaa gatctcctgt aagggttctg gctacaactt tagcaacaag 480tggatcggct gggtgcgcca attgcccggg agaggcctgg agtggatagc aatcatctat 540cccggttact cggacatcac ctacagcccg tccttccaag gccgcgtcac catctccgcc 600gacacgtcca ttaacaccgc ctacctgcac tggcacagcc tgaaggcctc ggacaccgcc 660atgtattatt gtgtgcgaca cacagctttg gccggctttg actactgggg cctgggcacc 720ctggtcaccg tctcctca 7387410PRTHomo sapiens 74Gly Phe Thr Phe Asp Asp Tyr Gly Met Ser1 5 107517PRTHomo sapiens 75Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Arg1 5 10 15Gly7612PRTHomo sapiens 76Glu Arg Gly Tyr Gly Tyr His Asp Pro His Asp Tyr1 5 107730DNAHomo sapiens 77gggttcacct ttgatgatta tggcatgagc 307851DNAHomo sapiens 78ggtattaatt ggaatggtgg tagcacaggt tatgcagact ctgtgagggg c 517936DNAHomo sapiens 79gagcgtggct acgggtacca tgatccccat gactac 368011PRTHomo sapiens 80Gly Arg Asn Asn Ile Gly Ser Lys Ser Val His1 5 10817PRTHomo sapiens 81Asp Asp Ser Asp Arg Pro Ser1 58211PRTHomo sapiens 82Gln Val Trp Asp Ser Ser Ser Asp His Val Val1 5 108333DNAHomo sapiens 83gggagaaaca acattggaag taaaagtgtg cac 338421DNAHomo sapiens 84gatgatagcg accggccctc a 218533DNAHomo sapiens 85caggtgtggg atagtagtag tgatcatgtg gta 3386121PRTHomo sapiens 86Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Arg Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Arg Glu Arg Gly Tyr Gly Tyr His Asp Pro His Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12087363DNAHomo sapiens 87gaagtgcagc tggtgcagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctgggtt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaggggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttgt attactgtgc gagagagcgt 300ggctacgggt accatgatcc ccatgactac tggggccaag gcaccctggt gaccgtctcc 360tca 36388109PRTHomo sapiens 88Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys1 5 10 15Thr Ala Arg Ile Thr Cys Gly Arg Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 10589327DNAHomo sapiens 89cagtctgtcg tgacgcagcc gccctcggtg tcagtggccc caggaaagac ggccaggatt 60acctgtggga gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc 300ggagggacca agctgaccgt cctaggt 32790249PRTHomo sapiens 90Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys1 5 10 15Thr Ala Arg Ile Thr Cys Gly Arg Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ser Arg Gly 100 105 110Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Leu Glu Met Ala 115 120 125Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Arg Pro Gly Gly 130 135 140Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr145 150 155 160Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 165 170 175Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val 180 185 190Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 195 200 205Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 210 215 220Ala Arg Glu Arg Gly Tyr Gly Tyr His Asp Pro His Asp Tyr Trp Gly225 230 235 240Gln Gly Thr Leu Val Thr Val Ser Ser 24591747DNAHomo sapiens 91cagtctgtcg tgacgcagcc gccctcggtg tcagtggccc caggaaagac ggccaggatt 60acctgtggga gaaacaacat tggaagtaaa agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtcgt ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240gatgaggccg actattactg tcaggtgtgg gatagtagta gtgatcatgt ggtattcggc 300ggagggacca agctgaccgt cctaggttct agaggtggtg gtggtagcgg cggcggcggc 360tctggtggat ccctcgagat ggccgaagtg cagctggtgc agtctggggg aggtgtggta 420cggcctgggg ggtccctgag actctcctgt gcagcctctg ggttcacctt tgatgattat 480ggcatgagct gggtccgcca agctccaggg aaggggctgg agtgggtctc tggtattaat 540tggaatggtg gtagcacagg ttatgcagac tctgtgaggg gccgattcac catctccaga 600gacaacgcca agaactccct gtatctgcaa atgaacagtc tgagagccga ggacacggcc 660ttgtattact gtgcgagaga gcgtggctac gggtaccatg atccccatga ctactggggc 720caaggcaccc tggtgaccgt ctcctca 7479210PRTHomo sapiens 92Gly Phe Ser Val Ser Gly Thr Tyr Met Gly1 5 109316PRTHomo sapiens 93Leu Leu Tyr Ser Gly Gly Gly Thr Tyr His Pro Ala Ser Leu Gln Gly1 5 10 159410PRTHomo sapiens 94Gly Gly Ala Gly Gly Gly His Phe Asp Ser1 5 109530DNAHomo sapiens 95gggttctccg tcagtggcac ctacatgggc 309648DNAHomo sapiens 96cttctttata gtggtggcgg cacataccac ccagcgtccc tgcagggc 489730DNAHomo sapiens 97ggaggggcag gaggtggcca ctttgactcc 309814PRTHomo sapiens 98Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His1 5 10997PRTHomo sapiens 99Gly Asn Ser Asn Arg Pro Ser1 510011PRTHomo sapiens 100Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val1 5 1010142DNAHomo sapiens 101actgggagca gctccaacat cggggcaggt tatgatgtac ac 4210221DNAHomo sapiens 102ggtaacagca atcggccctc a 2110333DNAHomo sapiens 103gcagcatggg atgacagcct gaatggttat gtc 33104118PRTHomo sapiens 104Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Leu Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Val Ser Gly Thr 20 25 30Tyr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Leu Leu Tyr Ser Gly Gly Gly Thr Tyr His Pro Ala Ser Leu Gln 50 55 60Gly Arg Phe Ile Val Ser Arg Asp Ser Ser Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys Gly Gly Ala Gly Gly Gly His Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser 115105354DNAHomo sapiens 105gaggtgcagc tggtggagac cggaggaggc ttgctccagc cgggggggtc cctcagactc 60tcctgtgcag cctctgggtt ctccgtcagt ggcacctaca tgggctgggt ccgccaggct 120ccagggaagg gactggagtg ggtcgcactt ctttatagtg gtggcggcac ataccaccca 180gcgtccctgc agggccgatt catcgtctcc agagacagct ccaagaatat ggtctatctt 240caaatgaata gcctgaaagc cgaggacacg gccgtctatt actgtgcgaa aggaggggca 300ggaggtggcc actttgactc ctggggccaa ggcaccctgg tgaccgtctc ctca 354106112PRTHomo sapiens 106Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40

45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu65 70 75 80Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95Leu Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly 100 105 110107336DNAHomo sapiens 107cagtctgtgt tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120cttccaggaa cagcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240cagtctgagg atgaggctga ttattactgt gcagcatggg atgacagcct gaatggttat 300gtcttcggaa ctgggaccaa gctgaccgtc ctaggt 336108251PRTHomo sapiens 108Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu65 70 75 80Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser 85 90 95Leu Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly 100 105 110Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125Ser Leu Glu Met Ala Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu 130 135 140Leu Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe145 150 155 160Ser Val Ser Gly Thr Tyr Met Gly Trp Val Arg Gln Ala Pro Gly Lys 165 170 175Gly Leu Glu Trp Val Ala Leu Leu Tyr Ser Gly Gly Gly Thr Tyr His 180 185 190Pro Ala Ser Leu Gln Gly Arg Phe Ile Val Ser Arg Asp Ser Ser Lys 195 200 205Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala 210 215 220Val Tyr Tyr Cys Ala Lys Gly Gly Ala Gly Gly Gly His Phe Asp Ser225 230 235 240Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250109753DNAHomo sapiens 109cagtctgtgt tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120cttccaggaa cagcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240cagtctgagg atgaggctga ttattactgt gcagcatggg atgacagcct gaatggttat 300gtcttcggaa ctgggaccaa gctgaccgtc ctaggttcta gaggtggtgg tggtagcggc 360ggcggcggct ctggtggtgg tggatccctc gagatggccg aggtgcagct ggtggagacc 420ggaggaggct tgctccagcc gggggggtcc ctcagactct cctgtgcagc ctctgggttc 480tccgtcagtg gcacctacat gggctgggtc cgccaggctc cagggaaggg actggagtgg 540gtcgcacttc tttatagtgg tggcggcaca taccacccag cgtccctgca gggccgattc 600atcgtctcca gagacagctc caagaatatg gtctatcttc aaatgaatag cctgaaagcc 660gaggacacgg ccgtctatta ctgtgcgaaa ggaggggcag gaggtggcca ctttgactcc 720tggggccaag gcaccctggt gaccgtctcc tca 7531109PRTHomo sapiens 110Ala Met Phe Pro Asn Ala Pro Tyr Leu1 51119PRTHomo sapiens 111Arg Met Ala Pro Asn Ala Pro Tyr Leu1 51129PRTHomo sapiens 112Arg Met Phe Ala Asn Ala Pro Tyr Leu1 51139PRTHomo sapiens 113Arg Met Phe Pro Ala Ala Pro Tyr Leu1 51149PRTHomo sapiens 114Arg Met Phe Pro Asn Ala Ala Tyr Leu1 51159PRTHomo sapiens 115Arg Met Phe Pro Asn Ala Pro Ala Leu1 51169PRTHomo sapiens 116Ile Leu Ser Leu Glu Leu Met Lys Leu1 51179PRTHomo sapiens 117Gln Leu Gln Asn Pro Ser Tyr Asp Lys1 5118449PRTHomo sapiens 118Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro1 5 10 15Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala 20 25 30Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr 35 40 45Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro 50 55 60Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly65 70 75 80Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe 85 90 95Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe 100 105 110Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe 115 120 125Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile 130 135 140Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr145 150 155 160Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe 165 170 175Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln 180 185 190Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser 195 200 205Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp 210 215 220Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln225 230 235 240Met Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser 245 250 255Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu 260 265 270Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile 275 280 285His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro 290 295 300Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys305 310 315 320Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys 325 330 335Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro 340 345 350Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp 355 360 365Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln 370 375 380Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr385 390 395 400His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys 405 410 415Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val 420 425 430Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu Gln Leu Ala 435 440 445Leu1197PRTHomo sapiens 119Ser Asn Ala Val Ala Trp Asn1 512013PRTHomo sapiens 120Arg Thr Tyr Arg Gly Ser Thr Tyr Tyr Ala Leu Ser Val1 5 101218PRTHomo sapiens 121Gly Ser Asn Ser Ala Phe Asp Phe1 51227PRTHomo sapiens 122Ser Asn Ser Ala Ala Trp Asn1 512316PRTHomo sapiens 123Arg Thr Tyr Tyr Gly Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10 151249PRTHomo sapiens 124Gly Arg Leu Gly Asp Ala Phe Asp Ile1 51257PRTHomo sapiens 125Ser Asp Gly Ala Ala Trp Asn1 512616PRTHomo sapiens 126Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10 151279PRTHomo sapiens 127Gly Asp Tyr Tyr Tyr Gly Met Asp Val1 51287PRTHomo sapiens 128Ser Asn Ala Ala Ala Trp Asn1 512916PRTHomo sapiens 129Arg Thr Tyr Tyr Gly Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5 10 151305PRTHomo sapiens 130Gly Ala Phe Asp Ile1 51315PRTHomo sapiens 131Ser Tyr Trp Ile Ser1 513217PRTHomo sapiens 132Arg Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe Gln1 5 10 15Gly1339PRTHomo sapiens 133Gly Asp Tyr Asp Phe Tyr Leu Asp Pro1 51345PRTHomo sapiens 134Ser Tyr Gly Ile Ser1 513517PRTHomo sapiens 135Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln1 5 10 15Gly13617PRTHomo sapiens 136Asp Leu Tyr Ser Ser Gly Trp Tyr Glu Ser Tyr Tyr Tyr Gly Met Asp1 5 10 15Val1375PRTHomo sapiens 137Ser Tyr Ala Ile Ser1 513817PRTHomo sapiens 138Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly13910PRTHomo sapiens 139Arg Ile Pro Pro Tyr Tyr Gly Met Asp Val1 5 1014017PRTHomo sapiens 140Trp Ile Ser Ala His Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln1 5 10 15Gly14110PRTHomo sapiens 141Asp Arg Val Trp Phe Gly Asp Leu Ser Asp1 5 1014217PRTHomo sapiens 142Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Glu Gln1 5 10 15Gly14312PRTHomo sapiens 143Asn Tyr Asp Phe Trp Ser Gly Asp Ala Phe Asp Ile1 5 1014412PRTHomo sapiens 144Ile Pro Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly1 5 101456PRTHomo sapiens 145Phe Tyr Gly Met Asp Val1 51465PRTHomo sapiens 146Asp Tyr Gly Met Ser1 514715PRTHomo sapiens 147Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val1 5 10 1514812PRTHomo sapiens 148Glu Arg Gly Tyr Gly Tyr His Asp Pro His Asp Tyr1 5 101495PRTHomo sapiens 149Asn Tyr Thr Met Asn1 515015PRTHomo sapiens 150Ser Ile Ser Leu Ser Gly Ala Tyr Ile Tyr Tyr Ala Asp Ser Leu1 5 10 1515113PRTHomo sapiens 151Glu Gly Tyr Ser Ser Ser Val Tyr Asp Ala Phe Asp Leu1 5 101525PRTHomo sapiens 152Ser Tyr Gly Met His1 515315PRTHomo sapiens 153Gly Ile Leu Ser Asp Gly Gly Lys Asp Tyr Tyr Val Asp Ser Val1 5 10 1515412PRTHomo sapiens 154Cys Ser Ser Asn Tyr Gly Asn Asp Ala Phe Asp Ile1 5 101555PRTHomo sapiens 155Thr Tyr Ser Met Asn1 515615PRTHomo sapiens 156Ser Ile Ser Ser Gly Ala Tyr Ser Ile Phe Tyr Ala Asp Ser Val1 5 10 1515713PRTHomo sapiens 157Asp Gln Tyr Tyr Gly Asp Lys Trp Asp Ala Phe Asp Ile1 5 101585PRTHomo sapiens 158Ser Tyr Gly Met Asn1 515914PRTHomo sapiens 159Ser Ile Ser Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val1 5 101609PRTHomo sapiens 160Glu Tyr Tyr Trp Asp Ala Phe Asp Ile1 516114PRTHomo sapiens 161Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn1 5 101628PRTHomo sapiens 162Ser Asn Asn Gln Arg Pro Ser Gly1 516313PRTHomo sapiens 163Ala Ala Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly1 5 1016413PRTHomo sapiens 164Glu Ala Trp Asp Asp Ser Leu Lys Gly Pro Val Phe Gly1 5 1016515PRTHomo sapiens 165Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His1 5 10 151668PRTHomo sapiens 166Gly Asn Ser Asn Arg Pro Ser Gly1 516715PRTHomo sapiens 167Gln Ser Tyr Asp Ser Ser Leu Ser Ala Asp Asn Tyr Val Phe Gly1 5 10 1516814PRTHomo sapiens 168Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg Asn Ile Val Asn1 5 101698PRTHomo sapiens 169Ser Asn Ile Glu Arg Pro Ser Gly1 517013PRTHomo sapiens 170Ala Ser Trp Asp Asp Ser Leu Asn Gly Val Leu Phe Gly1 5 1017114PRTHomo sapiens 171Cys Ser Gly Ser Arg Ser Asn Ile Ala Ser Asn Gly Val Gly1 5 101728PRTHomo sapiens 172Lys Asn Asp Gln Arg Pro Ser Gly1 517314PRTHomo sapiens 173Ser Ala Trp Asp Asp Ser Leu Asp Gly His Val Val Phe Gly1 5 1017413PRTHomo sapiens 174Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly1 5 1017514PRTHomo sapiens 175Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Ser Thr Val Asn1 5 101768PRTHomo sapiens 176Ser Asn Ser Gln Arg Pro Ser Gly1 517713PRTHomo sapiens 177Ala Ala Trp Asp Asp Ser Leu Asn Gly Val Val Phe Gly1 5 1017814PRTHomo sapiens 178Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr1 5 101798PRTHomo sapiens 179Arg Ser Asn Gln Arg Pro Ser Gly1 518014PRTHomo sapiens 180Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg Asn Thr Val Asn1 5 1018114PRTHomo sapiens 181Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asp Tyr Val Ser1 5 101828PRTHomo sapiens 182Asp Asn Asn Lys Arg Pro Ser Gly1 518313PRTHomo sapiens 183Gly Thr Trp Asp Asn Ser Leu Ser Ala Trp Val Phe Gly1 5 1018414PRTHomo sapiens 184Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Ser Val Tyr1 5 101858PRTHomo sapiens 185Asn Asn Asn Gln Arg Pro Ser Gly1 518613PRTHomo sapiens 186Ala Thr Trp Asp Asp Ser Leu Ser Gly Trp Val Phe Gly1 5 101878PRTHomo sapiens 187Arg Asn Asn Gln Arg Pro Ser Gly1 518813PRTHomo sapiens 188Ala Ala Trp Asp Asp Ser Leu Ser Ala Trp Val Phe Gly1 5 1018914PRTHomo sapiens 189Cys Ser Gly Ser Thr Ser Asn Ile Gly Ser Tyr Tyr Val Ser1 5 101908PRTHomo sapiens 190Asp Asn Asn Asn Arg Pro Ser Gly1 519113PRTHomo sapiens 191Gly Thr Trp Asp Ser Ser Leu Ser Ala Trp Val Phe Gly1 5 1019214PRTHomo sapiens 192Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser1 5 1019314PRTHomo sapiens 193Cys Ser Gly Ser Asn Ser Asn Ile Gly Thr Asn Thr Val Thr1 5 101948PRTHomo sapiens 194Ser Asn Phe Glu Arg Pro Ser Gly1 519513PRTHomo sapiens 195Ser Ala Trp Asp Asp Ser Phe Asn Gly Pro Val Phe Gly1 5 1019614PRTHomo sapiens 196Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Ser1 5 1019713PRTHomo sapiens 197Ala Ala Trp Asp Asp Gly Leu Arg Gly Tyr Val Phe Gly1 5 1019811PRTHomo sapiens 198Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 101997PRTHomo sapiens 199Ala Ala Ser Ser Leu Gln Ser1 52008PRTHomo sapiens 200Gln Gln Ser Tyr Ser Thr Pro Thr1 520111PRTHomo sapiens 201Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala1 5 102027PRTHomo sapiens 202Ala Ala Ser Thr Leu Gln Ser1 520310PRTHomo sapiens 203Gln Lys Tyr Asn Ser Ala Pro Gly Val Thr1 5 1020411PRTHomo sapiens 204Arg Ala Ser Gln Ser Ile Asn Gly Trp Leu Ala1 5 102057PRTHomo sapiens 205Arg Ala Ser Thr Leu Gln Ser1 52069PRTHomo sapiens 206Gln Gln Ser Ser Ser Leu Pro Phe Thr1 520711PRTHomo sapiens 207Arg Ala Ser Gln Gly Ile Ser Tyr Tyr Leu Ala1 5 102087PRTHomo sapiens 208Ala Ala Ser Thr Leu Lys Ser1 52099PRTHomo sapiens 209Gln Gln Leu Asn Ser Tyr Pro Leu Thr1 521011PRTHomo sapiens 210Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His1 5 102117PRTHomo sapiens 211Asp Asp Ser Asp Arg Pro Ser1 521211PRTHomo sapiens 212Gln Val Trp Asp Ser Ser Ser Asp His Pro Val1 5 1021311PRTHomo sapiens 213Gln Val Trp Asp Ser Ser Gly Asp His Pro Val1 5 102147PRTHomo sapiens 214Tyr Asp Ser Asp Arg Pro Ser1 521511PRTHomo sapiens 215Gly Gly Thr Asn Ile Gly Ser Arg Phe Val His1 5 1021611PRTHomo sapiens 216Gly Gly Asn Asn Val Glu Ser Lys Ser Val His1 5 102177PRTHomo sapiens 217Tyr Asp Arg Asp Arg Pro Ser1 521811PRTHomo sapiens 218Glu Val Trp Asp Ser Gly Ser Asp His Pro Val1 5 1021911PRTHomo sapiens 219Gly Gly Lys Asn Ile Gly Ser Lys Ser Val His1 5 1022011PRTHomo sapiens 220Gln Val Trp Asp Ser Gly Ser Asp His Tyr Val1 5 1022111PRTHomo sapiens 221Gln Val Trp Ile Ser Ser Gly Asp Arg Val Ile1 5 1022211PRTHomo sapiens 222Gly Gly Asp Asn Ile Gly Ser Gln Gly Val His1 5 102237PRTHomo sapiens 223Tyr Asp Thr Asp

Arg Pro Ser1 522411PRTHomo sapiens 224Gln Val Trp Gly Ala Ser Ser Asp His Pro Val1 5 1022514PRTHomo sapiens 225Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser1 5 102267PRTHomo sapiens 226Asp Val Ser Lys Arg Pro Ser1 522710PRTHomo sapiens 227Gly Ile Tyr Thr Tyr Ser Asp Ser Trp Val1 5 102287PRTHomo sapiens 228Asp Val Gly Asn Arg Pro Ser1 522910PRTHomo sapiens 229Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val1 5 1023014PRTHomo sapiens 230Thr Gly Thr Arg Ser Asp Val Gly Leu Tyr Asn Tyr Val Ala1 5 102317PRTHomo sapiens 231Asp Val Ile Tyr Arg Pro Gly1 523210PRTHomo sapiens 232Ser Ser Tyr Thr Asn Thr Gly Thr Val Leu1 5 1023314PRTHomo sapiens 233Thr Gly Thr Ser Ser Asp Phe Gly Asp Tyr Asp Tyr Val Ser1 5 102347PRTHomo sapiens 234Asp Val Ser Asp Arg Pro Ser1 523512PRTHomo sapiens 235Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Gly Val1 5 10236330PRTHomo sapiens 236Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330237106PRTHomo sapiens 237Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105238105PRTHomo sapiens 238Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val 35 40 45Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105

Claims

1. An isolated antibody, or antigen-binding fragment thereof, comprising one of: (A) (i) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; (ii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 20, 21 and 22; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 26, 27 and 28; (iii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 38, 39 and 40; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences selected from SEQ ID NOS: 44, 45 and 46; (iv) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 62, 63 and 64; (v) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 74, 75 and 76; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 80, 81 and 82; or (vi) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 92, 93 and 94; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 98, 99 and 100; or (B) a V.sub.H and V.sub.L comprising first and second amino acid sequences, respectively, selected from SEQ ID NOS: 14 and 16; 32 and 34; 50 and 52; 68 and 70; 86 and 88; and 104 and 106; or (C) an amino acid sequence selected from SEQ ID NOS: 18, 36, 54, 72, 90, and 108.

2. An isolated antibody, or antigen-binding fragment thereof, comprising: (A) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences selected from SEQ ID NOS: (i) 2, 3, and 4; (ii) 20, 21 and 22; (iii) 38, 39 and 40; (iv) 56, 57 and 58; (v) 74, 75 and 76; and (vi) 92, 93 and 94; and (B) a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences selected from SEQ ID NOS: (i) 8, 9 and 10; (ii) 26, 27 and 28; (iii) 44, 45 and 46; (iv) 62, 63 and 64; (v) 80, 81 and 82; and (vi) 98, 99 and 100.

3. An isolated antibody, or antigen-binding fragment thereof, comprising: a V.sub.H and V.sub.L comprising first and second amino acid sequences, respectively, selected from SEQ ID NOS: (i) 14 and 16; (ii) 32 and 34; (iii) 50 and 52; (iv) 68 and 70; (v) 86 and 88; and (vi) 104 and 106.

4. An isolated antibody, or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOS: 18, 36, 54, 72, 90, and 108.

5. The antibody of claim 1, wherein the antibody comprises a human variable region framework region.

6. The antibody of claim 1, wherein the antibody is fully human.

7. The antibody of claim 1, wherein said antibody specifically binds to an WT1 peptide bound to HLA-A2.

8. The antibody of claim 7, wherein said WT1 peptide has the amino acid sequence RMFPNAPYL (SEQ ID NO: 1).

9. The antibody of claim 7, wherein said HLA-A2 is HLA-A0201.

10. The antibody of claim 1, wherein the antigen-binding fragment of said antibody is an Fab, Fab', F(ab')2, Fv or single chain Fv (scFv).

11. The antibody of claim 1 conjugated to a therapeutic agent.

12. The antibody of claim 11, wherein said therapeutic agent is a drug, toxin, radioisotope, protein, or peptide.

13. An isolated nucleic acid that encodes an antibody of claim 1.

14. An isolated nucleic acid comprising: (A) (i) first, second and third nucleotide sequences selected from the group consisting of SEQ ID NOS: 5, 6 and 7; 23, 24 and 25; 41, 42 and 43; 58, 59 and 60; 77, 78 and 79; and 95, 96 and 97; and (ii) fourth, fifth and sixth nucleotide sequences selected from the group consisting of SEQ ID NOS: 11, 12 and 13; 29, 30 and 31; 47, 48 and 49; 65, 66 and 67; 83, 84 and 85; and 101, 102 and 103; or (B) first and second nucleotide sequences selected from the group consisting of SEQ ID NOS: 15 and 17; 33 and 35; 51 and 53; 69 and 71; 87 and 89; and 105 and 107; or (C) a nucleotide sequence selected from the group consisting of SEQ ID NOS: 19, 37, 55, 73, 91 and 109.

15. An isolated nucleic acid comprising: (A) first, second and third nucleotide sequences selected from the group consisting of SEQ ID NOS: (i) 5, 6 and 7; (ii) 23, 24 and 25; (iii) 41, 42 and 43; (iv) 58, 59 and 60; (v) 77, 78 and 79; and (vi) 95, 96 and 97; and (B) fourth, fifth and sixth nucleotide sequences selected from the group consisting of SEQ ID NOS: (vii) 11, 12 and 13; (viii) 29, 30 and 31; (ix) 47, 48 and 49; (x) 65, 66 and 67; (xi) 83, 84 and 85; and (xii) 101, 102 and 103.

16. The nucleic acid of claim 15, wherein said first, second and third nucleotide sequences encode HC-CDR1, HC-CDR2 and HC-CDR3, respectively of an antibody and said fourth, fifth and sixth nucleotide sequences encodes LC-CDR1, LC-CDR2 and LC-CDR3, respectively of said antibody.

17. An isolated nucleic acid comprising first and second nucleotide sequences respectively, selected from the group consisting of SEQ ID NOS: (i) 15 and 17; (ii) 33 and 35; (iii) 51 and 53; (iv) 69 and 71; (v) 87 and 89 and (vi) 105 and 107.

18. The nucleic acid of claim 17, wherein said first nucleotide sequence encodes a VH, and said second nucleotide sequence encodes a VL of an antibody.

19. An isolated nucleic acid, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 19, 37, 55, 73, 91 and 109.

20. The nucleic acid of claim 19, wherein said nucleic acid encodes an scFv.

21. A vector comprising a nucleic acid of claim 13.

22. A cell comprising a nucleic acid of claim 13.

23. The cell of claim 22, wherein said cell expresses on its surface an antibody of claim 1.

24. A method for detecting WT1 in a whole cell or tissue, said method comprising: (A) contacting a cell or tissue with an antibody or antigen-binding fragment thereof that specifically binds to a WT1 peptide that is bound to HLA-A2, said antibody or antigen-binding fragment having a K.sub.D value of 1.17.times.10.sup.-8M or lower, and wherein said antibody comprises a detectable label; (B) determining the amount of antibody bound to said cell or tissue by measuring the amount of detectable label associated with said cell or tissue, wherein the amount of bound antibody indicates the mount of WT1 in said cell or tissue.

25. The method of claim 24, wherein the antibody specifically binds to a WT1 peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1), bound to HLA-A2, optionally wherein the WT1 peptide is bound to HLA-A0201.

26. The method of claim 24, wherein the antibody or antigen-binding fragment has a K.sub.D value ranging from 1.17.times.10.sup.-8 M to 7.68.times.10.sup.-9 M.

27. The method of claim 24, wherein the antibody is an isolated antibody or antigen-binding fragment thereof comprising one of: (A) (i) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 20, 21 and 22; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 26, 27 and 28; (ii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 62, 63 and 64; (B) a V.sub.H and V.sub.L comprising first and second amino acid sequences, respectively, selected from SEQ ID NOS: 32 and 34; 68 and 70; or (C) an amino acid sequence selected from SEQ ID NOS: 36, and 72.

28. A kit comprising an antibody of claim 1.

29. (canceled)

30. A pharmaceutical composition comprising an antibody of claim 1 and a pharmaceutically acceptable carrier.

31. A method for treatment of a subject having a WT1-positive disease, comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds to a WT1 peptide that is bound to HLA-A2, said antibody or antigen-binding fragment having a K.sub.D value of 1.17.times.10.sup.-8 M or lower.

32. The method of claim 31, wherein the WT1-positive disease is a chronic leukemia or acute leukemia or WT1.sup.+ cancer.

33. The method of claim 31, wherein the WT1-positive disease is selected from the group consisting of chronic myelocytic leukemia, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma.

34. The method of claim 31, wherein said antibody is a conjugate having a cytotoxic moiety linked thereto.

35. A method for treatment of a subject having a WT1-positive disease, comprising administering to the subject a therapeutically effective amount of a nucleic acid that encodes an antibody or antigen-binding fragment thereof that specifically binds to a WT1 peptide that is bound to HLA-A2, said antibody or antigen-binding fragment having a K.sub.D value of 1.17.times.10.sup.-8 M or lower.

36. The method of claim 35, wherein the WT1-positive disease is a chronic leukemia, acute leukemia or WT.sup.+ cancer.

37. The method of claim 35, wherein the WT1-positive disease is selected from the group consisting of chronic myelocytic leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma.

38. (canceled)

39. A pharmaceutical composition comprising a nucleic acid of claim 13.

40. A method for killing a WT1.sup.+ cell, said method comprising: contacting the cell with an antibody or antigen-binding fragment thereof that specifically binds to a WT1 peptide that is bound to HLA-A2, said antibody or antigen-binding fragment having a K.sub.D value of 1.17.times.10.sup.-8 M or lower.

41. The method of claim 40, wherein said antibody or antigen-binding fragment thereof has a cytotoxic moiety linked thereto.

42. The method of any one of claims 31, 35 and 40, wherein the antibody is an isolated antibody or antigen-binding fragment thereof comprising one of: (A) (i) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 20, 21 and 22; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 26, 27 and 28; or (ii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 62, 63 and 64; or (B) a V.sub.H and V.sub.L comprising first and second amino acid sequences, respectively, selected from SEQ ID NOS: 32 and 34; and 68 and 70; or (C) an amino acid sequence selected from SEQ ID NOS: 36, and 72.

43. The method of any one of claims 31, 35 and 40, wherein the antibody or antigen-binding fragment has a K.sub.D value ranging from 1.17.times.10.sup.-8 M to 7.68.times.10.sup.-9 M.