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AU2014228145A1 - Combination/adjuvant therapy for WT-1-positive disease - Google Patents

Combination/adjuvant therapy for WT-1-positive disease Download PDF

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AU2014228145A1
AU2014228145A1 AU2014228145A AU2014228145A AU2014228145A1 AU 2014228145 A1 AU2014228145 A1 AU 2014228145A1 AU 2014228145 A AU2014228145 A AU 2014228145A AU 2014228145 A AU2014228145 A AU 2014228145A AU 2014228145 A1 AU2014228145 A1 AU 2014228145A1
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Leonid DUBROVSKY
David Scheinberg
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Memorial Sloan Kettering Cancer Center
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Abstract

In an attempt to improve primary disease responsiveness and/or to overcome resistant disease, the present disclosure provides a method for treating or inhibiting the proliferation of a WT-1 -dependent cancer comprising providing to a subject in need thereof a therapeutically effective amount of a tyrosine kinase inhibitor along with an anti-WT-1 /HLA antibody, that is, an antibody that specifically binds to a peptide of Wilms' tumor protein (WT-1 ) presented on the surface of the cancer cells in an HLA-restricted fashion.

Description

WO 2014/143835 PCT/US2014/027977 -1 COMBINATION/ADJUVANT THERAPY FOR WT-1 -POSITIVE DISEASE Cross-Reference to Related Applications [0001] This application contains subject matter that is related to the subject matter of commonly-assigned PCT international application serial no. PCT/US2012/031892 filed on April 2, 2012 entitled "T-Cell Receptor Like Antibodies Specific for WT1 Peptides " and commonly assigned, US provisional application no. 61/798,563, entitled "Antibodies to Cytosolic Peptides" (Attorney Docket No.3314.030P) filed March 15, 2013; the contents of each are hereby incorporated herein by reference in their entirety. [0002] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. 61/794,168 filed March 15, 2013, and U.S. Provisional Patent Application Serial No. 61/880,585 filed September 20, 2013, is hereby claimed, and the disclosures of both priority documents are incorporated herein by reference in their entirety. Statement of Rights Under Federally-Sponsored Research [0003] This invention was made with government support under grant NIH RO1 CA 55349 and P01 CA 23766 awarded by the U.S. National Institutes of Health. The government has certain rights in the invention. Sequence Listinq [0004] This application contains a Sequence Listing, created on March 14, 2014; the file, in ASCII format, is designated 48319_SeqListing.txt and is 182,226 bytes. The file is hereby incorporated by reference in its entirety into the application. Technical Field [0005] The present invention relates generally to a treatment for WT-1 -positive diseases like chronic myelogenous leukemia (CML). More particularly, the invention relates to inhibition of tumor growth and combination treatment with a tyrosine kinase WO 2014/143835 PCT/US2014/027977 -2 inhibitor therapeutic agent and antibodies against Wilm's tumor oncogene protein (WT-1). Background of the Invention [0006] To date, the treatment of cancers like CML has relied on therapeutic agents that target protein tyrosine kinase. Tyrosine kinase inhibitors (TKIs) include imatinib (GLEEVEC*) dasatinib (SPRYCEL*), sunitinib, sorafenib, pazopanib, to name a few. Tyrosine kinase inhibitors are currently the first line therapeutic in the treatment of chronic myelogenous (also referred to as myeloid or myelocytic) leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS), ovarian cancer, prostate cancer, soft tissue sarcoma, malignant glioma, renal cell cancer, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), breast cancer, lung cancer etc. However, the clinical efficacy of some TKIs, for example, imatinib and sunitinib, are limited by rare patient-specific intolerance to the drug or the development of treatment-refractory disease. [0007] In addition to small molecule therapeutics that target the tyrosine kinase protein, treatments of leukemia based on immunologic approaches using vaccines and tumor-specific antibodies are being developed. For example, the Wilms' tumor oncogene protein (WT-1) has become an attractive target for immunotherapy for most leukemias, including CML, and a wide range of cancers. WT-1 is a zinc finger transcription factor that is normally expressed in mesodermal tissues during embryogenesis. In normal adult tissue, WT-1 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, WT-1 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 WT-1 are detected in patients with hematopoietic malignancies and solid tumors, indicating that WT-1 is a highly immunogenic antigen (7).
WO 2014/143835 PCT/US2014/027977 -3 [0008] For the most part, clinically approved therapeutic monoclonal antibodies (mAbs) recognize structures of cell surface proteins. WT-1, however, is a nuclear protein and, therefore, is inaccessible to classical antibody therapy. Until recently, immunotherapy targeting WT-1 had been limited to cellular approaches, exclusively aimed at generating WT-1 -specific cytotoxic CD8 T cell (CTL) responses that recognize peptides presented on the cell surface by MHC class I molecules. [0009] 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 WT-1 protein induce HLA-restricted cytotoxic CD8 T cells, which are capable of killing tumor cells. [0010] Other approaches to cancer treatment target cancer antigens 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. [0011] A tremendous benefit would exist if there existed an adjuvant therapeutic approach that would improve primary disease responsiveness, overcome resistant disease, and/or lower the effective dose of an individual therapeutic agent, for example, to avoid toxicity and other adverse side effects of the TKI. Summary of the Invention [0012] The present disclosure provides a method for the treatment of WT-1 positive diseases based on a combination of therapeutic agents directed to different molecular targets. The approach incorporates conventional treatment with tyrosine kinase inhibitors (TKIs) such as those directed at Bcr-Abl, (imatinib and dasatinib), and TKIs directed to other molecular targets such as EGFR, for example, erlotinib and gefitinib as well as an immunotherapeutic approach based on the administration WO 2014/143835 PCT/US2014/027977 -4 of antibodies that recognize and bind to peptides of WT-1 oncoprotein in an HLA restricted fashion. [0013] The present invention is based on the unexpected observation that a treatment regimen that combines a TKI and an anti-WT-1 antibody results in earlier inhibition of tumor growth and an improved anti-tumor response when compared to either administered individually. In some embodiments, co-administration of TKI with anti-WT-1 antibody permits the use of amounts of TKI that are lower than those currently utilized in treating the above-identified conditions, while maintaining the therapeutic efficacy of the TKI and moreover, while improving time-to-tumor progression, overall survival and decreasing TKI-associated side effects. [0014] In one aspect, therefore, the invention relates to a method for treating or inhibiting the growth of a WT-1 -positive cancer in a subject by administering a therapeutically effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an isolated anti WT-1 antibody, or antigen-binding portion thereof, that is, an antibody that specifically binds to a WT-1 peptide bound to an MHC antigen. The tyrosine kinase inhibitor may be directed to a molecular target such as Bcr-Abl (imatinib, dasatinib and nilotinib), EGFR (erlotinib and gefitinib), VEGFR-1 (pazopanib and sorafenib) and others. [0015] In one aspect, the WT-1 positive cancer is selected from the group consisting of chronic myelogenous leukemia (CML), 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, gastrointestinal stromal tumors (GIST) and others including solid tumors. [0016] In one aspect, the tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, pazopanib and sunitinib. In one embodiment, the tyrosine kinase inhibitor is imatinib or dasatinib or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutically acceptable salt of imatinib is imatinib mesylate.
WO 2014/143835 PCT/US2014/027977 -5 [0017] In another aspect, the invention relates to combination/adjuvant therapy with a TKI and an isolated anti-WT-1 antibody, or antigen-binding portion thereof. Examples of anti-WT-1 antibodies for use in combination therapy with a TKI include but are not limited to: [0018] an anti-WT-1 antibody comprising a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 comprising amino acid sequences as shown in Tables 1 to 14 below and Figures 7-10. [0019] In another aspect, the WT-1 antibody, or antigen-binding fragment thereof, comprises a VH and VL comprising first and second amino acid sequences, as shown in Tables 1 to 14 below and Figures 7-10. In yet another aspect, the WT-1 antibody comprises the amino acid sequence of an scFv shown in Tables 1 to 14 below and Figures 7-10. [0020] The disclosed method employs a WT-1 antibody that is fully human; the antibody comprises a human variable region framework region and human constant regions. The WT-1 antibody specifically binds to a WT-1 peptide in an HLA restricted manner with a K 0 less than 1 X 10- 8 M; in one embodiment, the K 0 is in the range of about 1 X 10 11 M to about 1 X 10- 8 M. The WT-1 antibody induces antibody dependent cellular cytotoxicity (ADCC) against WT-1 -positive cells. Brief Description of the Drawings [0021] Figure 1 shows that imatinib at 1 pM, 5 pM or 10 pM does not affect antigen dependent cellular cytotoxicity of human effector cells at various effector: target ratios (E:T) against fresh BV1 73 cells, a leukemia cell line derived from CML in blastic crisis (HLA-A0201 *, Philadelphia chromosome positive). Effector to target ratios varied to demonstrate the dependence of ESKM ADCC on high E:T ratios. [0022] Figure 2 shows the effect of an anti-WT-1/HLA-A antibody, designated ESKM, with and without imatinib on tumor growth at intervals of 13, 20, 27, 34 and 40 days.
WO 2014/143835 PCT/US2014/027977 -6 [0023] Figure 3 shows luciferin imaging of BV1 73 xenograft NSG mice after 5 weeks of daily administration of 50mg/kg imatinib with (lower right panel) and without (lower left panel) administration of 100 pg anti-WT-1/HLA-A antibody twice a week to mice with tumors. Control animals received neither imatinib nor antibody (upper left). [0024] Figure 4 shows the effects of administration of ESKM and dasatinib at 1 PM, 5 pM or 10 pM on ADCC of human effectors cells at various effector: target ratios (E:T) against BV1 73. [0025] Figure 5 shows the effect of treatment with dasatinib alone or in combination with an anti-WT-1 antibody on BV1 73 tumor growth in NSG mice over four weeks of treatment. [0026] Figure 6 shows luciferin imaging of BV1 73 xenograft NSG mice after five weeks of therapy with a control treatment with dasatinib alone or in combination with an anti-WT-1 antibody. [0027] Figures 7-10 show amino acid sequences, including consensus sequences, for the CDRs of some embodiments of anti-WT-1 antibodies useful for the combination therapy of the disclosure. [0028] Figure 11 shows the growth of BV1 73R following start of therapy on day 6 with tyrosine kinase inhibitors, Imatinib and Dasatinib, and anti-WT-1/HLA0201 antibody (ESKM). 0 control; E ESKM only; A Imatinib only; X Imatinib and ESKM; >< Dasatinib only; 0 Dasatinib and ESKM. [0029] Figure 12 shows the growth of BV173R in NSG mice treated with ESKM and Ponatinib: o control; A ESKM only; X Ponatinib only; >< Ponatinib and ESKM. [0030] Figure 13 shows luciferin imaging of BV173 xenograft NSG mice after five weeks of therapy. Detailed Description of the Invention [0031] All publications, patents and other references cited herein are incorporated by reference in their entirety into the present disclosure. Subject matter incorporated WO 2014/143835 PCT/US2014/027977 -7 by reference is not considered to be an alternative to any claim limitations, unless otherwise explicitly indicated. [0032] In practicing the present invention, many conventional techniques in immunology are used, which are within the skill of the ordinary artisan. These techniques are described in greater detail in, for example, "Current Protocols in Immunology" (John E. Coligan et al., eds., John Wiley & Sons, Inc. 1991 and periodic updates); Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009. 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 and dosage information are hereby incorporated by reference as part of the present disclosure. The following abbreviations are used throughout the application: [0033] Ab: Antibody [0034] ADCC: Antibody-dependent cellular cytotoxicity [0035] ALL: Acute lymphocytic leukemia [0036] AML: Acute myeloid leukemia [0037] CDC: Complement dependent cytotoxicity [0038] CMC: Complement mediated cytotoxicity [0039] CDR: Complementarity determining region (see also HVR below) [0040] CL: Constant domain of the light chain [0041] CH 1 : 1 st constant domain of the heavy chain [0042] CH 1 , 2, 3: 1 st, 2 nd and 3 rd constant domains of the heavy chain [00431 CH 2 , 3: 2and rd constant domains of the heavy chain [0044] CHO: Chinese hamster ovary WO 2014/143835 PCT/US2014/027977 -8 [0045] CML: chronic myelogenous leukemia; also referred to as chronic myelocytic leukemia and chronic myeloid leukemia [0046] CTL: Cytotoxic T cell [0047] E:T Ratio: Effector:Target ratio [0048] Fab: Antibody binding fragment [0049] FACS: Fluorescence-activated cell sorting [0050] FBS: Fetal bovine serum [0051] FR: Framework region [0052] HC: Heavy chain [0053] HLA: Human leukocyte antigen [0054] HVR-H: Hypervariable region-heavy chain (see also CDR) [0055] HVR-L: Hypervariable region-light chain (see also CDR) [0056] Ig: Immunoglobulin [0057] KD: Dissociation constant [0058] ko 0 i: Dissociation rate [0059] kon: Association rate [0060] MHC: Major histocompatibility complex [0061] MM: Multiple myeloma [0062] scFv: Single-chain variable fragment [0063] TKI: tyrosine kinase inhibitor [0064] VH: Variable heavy chain includes heavy chain hypervariable region and heavy chain variable framework region WO 2014/143835 PCT/US2014/027977 -9 [0065] VL: Variable light chain includes light chain hypervariable region and light chain variable framework region [0066] WT-1: Wilms tumor protein 1 [0067] In the description that follows, 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. The definitions provided herein below are meant to clarify, but not limit, the terms defined. [0068] As used herein, "administering" and "administration" refer to the application of an active ingredient to the body of a subject. [0069] "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 VH) 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 VL) and a light chain constant CL region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. 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 WO 2014/143835 PCT/US2014/027977 -10 system (e.g., effector cells) and the first component (Clq) of the classical complement system. [0070] 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 VL, VH, CL and CH1 domains; a F(ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). [0071] Furthermore, although the two domains of the Fv fragment, VLand VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. 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. NatI. 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. [0072] An "isolated antibody" is intended to encompass antibodies which have been identified and separated and/or recovered from a component of its natural environment as well as "synthetic antibodies" or "recombinant antibodies," antibodies that are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art.
WO 2014/143835 PCT/US2014/027977 -11 [0073] As used herein, the term "effective amount" means that amount of a compound or therapeutic agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. [0074] The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. [0075] The present invention provides an improved treatment method for WT-1 positive disease by the co-administration of a tyrosine kinase inhibitor and an anti WT-1 antibody. Tyrosine Kinase Inhibitors [0076] Tyrosine kinase inhibitors, as well as routes of administration and appropriate dose considerations are well known in the art for the treatment of certain cancers. These small-molecule drugs target several members of a class of proteins called tyrosine kinase enzymes that participate in signal transduction. These enzymes are overactive in some cancers, leading to uncontrolled growth. [0077] Tyrosine kinase inhibitors suitable for use in the disclosed method include imatinib, dasatinib, nilotinib, bosutinib, ponatinib, and bafetinib imatinib, dasatinib, nilotinib, bosutinib, ponatinib, and bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib. The table below provides a list of some TKIs, their molecular targets, and FDA-approved indications. Table Drug FDA-Approved Toxicities, Side Effects (Trade Target Indications and Precautions Monitoring name) Dasatnib CR- Chronic myeloid Rash; diarrhea; pleural CBC: EKG: LFTs: aSrveel) AIBL, SRC leukemia, acute effusion; fluid retention; weight; signs and (Sprycel) fan.........................y, c.......... lyrnphocyti ........................ sym ptom s of fluid..............................
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'.................... ... ............................................................................................................................ : WO 2014/143835 PCT/US2014/027977 -13 amylase and lipase levels; myelosuppression; wound-healing complications; need to discontinue treatment temporarily for surgical procedures Adrenal function in Nasaand vomiting; patients with trauma yellow discoloration of inr tsere undergingo skin; hypothyroidism:;i sursery bleroing depression of LVEF:; ugry lo Suntiib FR, Renal cell cancer, adrenal function prsue EKG; (Sutent) c-KlT, gastrointestinal abnormalities; diarrhea; eLeF;olt level (Sutnt)c-KT, stromal tumor myelosuppression: electrosium level mucositis:;lvtdlps potassium); and creatinine levels; phosphate levels; elevated LFTs; increased sgsadsmtm uric acid levels sinn smtm of pancreatitis; thyroid function tests [0078] Imatinib mesylate (marketed as GLEEVEC@) is approved to treat gastrointestinal stromal tumor (GIST, a rare cancer of the gastrointestinal tract) and other mesenchymal tumors, Ph* CML, certain other kinds of leukemia, dermatofibrosarcoma protuberans, myelodysplastic/myeloproliferative disorders, and systemic mastocytosis. Imatinib is generally regarded as the first generation of Bcr Abl tyrosine kinase inhibitors used for the treatment of, for example, CML. The GLEEVEC@ Prescribing Information [2013: Novartis] (which is incorporated by reference in its entirety), lists recommendations for imatinib administration and relevant data. [0079] Dasatinib (marketed as SPRYCEL@) is approved to treat some patients with CML or acute lymphoblastic leukemia. The drug is a small-molecule inhibitor of several tyrosine kinase enzymes. The SPRYCEL@ Prescribing Information [Bristol Myers Squibb] (which is incorporated by reference in its entirety), lists recommendations for dasatinib administration and relevant data.
WO 2014/143835 PCT/US2014/027977 -14 [0080] Nilotinib (marketed as TASIGNA@) is approved to treat some patients with CML. The drug is another small-molecule tyrosine kinase inhibitor. The TASIGNA@ Prescribing Information [Novartis] (which is incorporated by reference in its entirety), lists recommendations for nilotinib administration and relevant data. [0081] Bosutinib (marketed as BOSULIF®) is also approved to treat some patients with CML and is another example of a small-molecule tyrosine kinase inhibitor. The BOSULIF@ Prescribing Information [Pfizer] (which is incorporated by reference in its entirety), lists recommendations for bosutinib administration and relevant data. [0082] Ponatinib (marketed as ICLUSIGTM) is an FDA approved oral drug candidate for the treatment of CML and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Ponatinib is a multi-targeted TKI, although the primary target for ponatinib is BCR-ABL, an abnormal tyrosine kinase that is the hallmark of CML and Ph+ ALL. [0083] Prescribing Information for each of the therapeutic agents listed herein is hereby incorporated by reference in its entirety. Additional information regarding dosing and/or adverse side effects of tyrosine kinase inhibitors can be found in G.D. Demetri, Differential properties of current tyrosine kinase inhibitors in gastrointestinal stromal tumors; Warnault P et al. Recent Advances in Drug Design of Epidermal Growth Factor Receptor Inhibitors; Sivendran S et al. Treatment-related mortality with vascular endothelial growth factor receptor tyrosine kinase inhibitor therapy in patients with advanced solid tumors: a meta-analysis; Cabez6n-Guti6rrez L. ALK mutated non-small-cell lung cancer: a new strategy for cancer treatment; Barni, S. The risk for anemia with targeted therapies for solid tumor; Dasanu, CA Cardiovascular toxicity associated with small molecule tyrosine kinase inhibitors currently in clinical use. (See reference nos. 69-74 below) Anti-WT-1 Antibodies [0084] The Wilms' tumor oncogene protein (WT-1) is an attractive target for immunotherapy for most leukemias and a wide range of cancers. WT-1 is a zinc finger transcription factor that is normally expressed in mesodermal tissues during embryogenesis. In normal adult tissue, WT-1 expression is limited to low levels in WO 2014/143835 PCT/US2014/027977 -15 CD34* hematopoietic stem cells but is over-expressed in leukemias of multiple lineages and a wide range of solid tumors (1-2). More recently, WT-1 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 WT-1 are detected in patients with hematopoietic malignancies and solid tumors, indicating that WT-1 is a highly immunogenic antigen (7). [0085] For the most part, clinically approved therapeutic monoclonal antibodies (mAbs) (for example, trastuzumab) recognize structures of cell surface proteins. WT 1, however, is a nuclear protein and, therefore, is inaccessible to classical antibody therapy. Until recently, immunotherapy targeting WT-1 has been limited to cellular approaches, exclusively aimed at generating WT-1 -specific cytotoxic CD8 T cell (CTL) responses that recognize peptides presented on the cell surface by MHC class I molecules. [0086] 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 WT-1 protein induce HLA-restricted cytotoxic CD8 T cells, which are capable of killing tumor cells. [0087] It has now been determined that co-administration of anti-WT-1 antibodies with a small molecule tyrosine kinase inhibitor can enhance the efficacy of the small molecule therapeutic. [0088] Anti-WT-1 antibodies that may be of use for combination therapy of cancer within the scope of the claimed methods and compositions include, but are not limited to those anti-WT-1 antibodies that specifically bind a WT-1 peptide in an HLA restricted manner and further exhibit at least one of the following properties: (a) binds to WT-1/HLA with a K 0 of about 1X1 0 11 M to 1X10 8 M; (b) induces antibody dependent cellular cytotoxicity (ADCC) against WT-1 -expressing cells; or (c) inhibits WO 2014/143835 PCT/US2014/027977 -16 growth of WT-1 positive cells in vivo. In some embodiments, anti-WT-1 antibodies to be paired with TKI administration are those comprising one or more amino acid sequences (scFv, VH and VL regions or CDRs) listed in Tables 1-14 and shown in Figures 7-10. Table 1 WT-1 Antigen 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) tctggaagcagctccaac aggagtaatcagcggccctca gcagcatgggatgacagcctg DNA atcggaagtaattatgtat (SEQ ID NO: 12) aatggtgtggta ac (SEQ ID NO: 11) 1 (SEQ ID NO: 13) Full QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE VH WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY CARRIPPYYGMDVWGQGTTVTVSS (SEQ ID NO: 14) DNA caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg cagagtcacgattaccg cgg acg aatccacgag cacagcctacatg gag ctgag cagcctgag 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) WO 2014/143835 PCT/US2014/027977 -17 WT-1 Antigen Peptide RMFPNAPYL (SEQ ID NO: 1) 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) Table 2 WT-1 (Ext002 #5) Antigen 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) WO 2014/143835 PCT/US2014/027977 -18 WT-1 (Ext002 #5) Antigen Peptide RMFPNAPYL (SEQ ID NO: 1) 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 gacatccag atgacccagtctccatcctccctgtctgcatctgtag gag acagagtcaccatcacttg ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta agotcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca gagttacagtacccctctcactttcgg cg gag g g accaaagtgg atatcaaacgt (SEQ ID NO: 35) scFv DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT FGGGTKVDIKRSRGGGGSGGGGSGGGGSLEMAQVQLQQSGPGLVK PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWY NDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGRLGDAF DIWGQGTMVTVSS (SEQ ID NO: 36) DNA gacatccag atgacccagtctccatcctccctgtctgcatctgtag gag acagagtcaccatcacttg ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta agotcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca gagttacagtacccctctcactttcgg cg gag g g accaaagtgg atatcaaacgttctagaggtg gtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtac agctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatct ccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggc cttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctgtgaaaa gtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactc ccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctggggccaa gggacaatggtcaccgtctcttca (SEQ ID NO: 37) WO 2014/143835 PCT/US2014/027977 -19 Table 3 WT-1 (Ext002 #13) Antigen 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: 43) (SEQ ID NO: 42) 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) 1 (SEQ ID NO: 49) Full VH QMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLE WMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTA MYYCARVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 50) DNA cagatg cagctg gtgcagtccg gag cag ag gtg aaagag cccgggg agtctctgag g atctcct gtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaa gg cctg gagtg g atgg g gag ggttgatcctg gctactcttatag cacctacagcccgtccttccaag gccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaag gcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacc cctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 51) Full QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK VL LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGWVFGGGTKLTVLG (SEQ ID NO: 52) DNA caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt tctggaagcagotccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggt (SEQ ID NO: 53) scFv QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGWVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQMQLV QSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLEWMGR VDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCA RVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 54) WO 2014/143835 PCT/US2014/027977 -20 WT-1 (Ext002 #13) Antigen Peptide RMFPNAPYL (SEQ ID NO: 1) DNA caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt tctggaagcagotccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatgg cccagatg cagctg gtgcagtccg gag cag ag gtg aaagag cccgggg agtctctg ag gatct cctgtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccggga aaggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttcca aggccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctga aggcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcga cccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 55) Table 4 WT-1 (Ext002 #15) Antigen 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) 1_1 Full QVQLVQSGAEVKKPGESLKISCKGSGYNFSNKWIGWVRQLPGRGLE VH WIAIIYPGYSDITYSPSFQGRVTISADTSINTAYLHWHSLKASDTAMYYC VRHTALAGFDYWGLGTLVTVSS (SEQ ID NO: 68) DNA caggtgcagctg gtgcagtctg gag cag ag gtg aaaaagcccg gag agtctctgaag atctcctg taagg gttctg gctacaactttagcaacaagtgg atcgg ctg ggtg cgccaattg cccgg gag ag g cctggagtggatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggc cgcgtcaccatctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggc ctcggacaccgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgg gcaccctggtcaccgtctcctca (SEQ ID NO: 69) Full DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI VL YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT FGQGTKVEIKR (SEQ ID NO: 70) WO 2014/143835 PCT/US2014/027977 -21 WT-1 (Ext002 #15) Antigen Peptide RMFPNAPYL (SEQ ID NO: 1) DNA gacatccag atgacccagtctccttccaccctgtctgcatctgtag gag acagagtcacaatcacttg ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct cagotcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO: 71) scFv DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT FGQGTKVEIKRSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKK PGESLKISCKGSGYNFSNKWIGWVRQLPGRGLEWIAIIYPGYSDITYSP SFQGRVTISADTSINTAYLHWHSLKASDTAMYYCVRHTALAGFDYWGL GTLVTVSS (SEQ ID NO: 72) DNA gacatccag atgacccagtctccttccaccctgtctgcatctgtag gag acagagtcacaatcacttg ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct cagotcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgttctagaggtggt ggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtgcag ctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctgtaagggttct ggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagaggcctggagtg gatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggccgcgtcacc atctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggcctcggacac cgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgggcaccctggt caccgtctcctca (SEQ ID NO: 73) Table 5 WT-1 (Ext002 #18) Antigen 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) WO 2014/143835 PCT/US2014/027977 -22 WT-1 (Ext002 #18) Antigen Peptide RMFPNAPYL (SEQ ID NO: 1) DNA gggagaaacaacattgg gatgatagcgaccggccctc caggtgtgggatagtagtagt aagtaaaagtgtgcac a gatcatgtggta (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85) Full EVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKG VH LEWVSGINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAE DTALYYCARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 86) DNA gaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtccctgagactctcct gtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagotccagggaag gggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgagg ggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctg agagccgaggacacggccttgtattactgtgcgagagcaggtggctacgggtaccatgatccc catgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 87) Full QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL VL VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVLG (SEQ ID NO: 88) DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc gactattactgtcag gtgtgg g atagtagtagtgatcatgtggtattcgg cg gag g g accaag ct gaccgtcctaggt (SEQ ID NO: 89) scFv QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVLGSRGGGGSGGGGSGGSLEMAEVQLVQ SGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVS GINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTALYY CARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 90) DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc gactattactgtcag gtgtgg g atagtagtagtgatcatgtggtattcgg cg gag g g accaag ct gaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggatccc tcgagatggccgaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtcc ctgagactctcctgtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaag ctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgca gactctgtgaggggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaa atgaacagtctgagagccgaggacacggccttgtattactgtgcgagagagcgtggctacggg taccatgatccccatgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 91) WO 2014/143835 PCT/US2014/027977 -23 Table 6 WT-1 (Ext002 #23) Antigen Peptide RMFPNAPYL (SEQ ID NO. 1) CDRs 1 2 3 VH GFSVSGTYMG LLYSGGGTYHPASLQ GGAGGGHFDS (SEQ ID NO. 92) G (SEQ ID NO. 94) (SEQ ID NO. 93) DNA gggttctccgtcagtggcac cttctttatagtggtggcggcac gaggggcaggaggtggcc ctacatgggc(SEQ ID ataccacccagcgtccctgca actttgactcc (SEQ ID NO. 95) 999c NO. 97) (SEQ ID NO. 96) 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. 103) (SEQ ID NO. 101) Full EVQLVETGGGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLE VH WVALLYSGGGTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVY YCAKGGAGGGHFDSWGQGTLVTVSS (SEQ ID NO. 104) DNA gaggtgcag ctggtggagaccggaggagg cttgctccag ccgggggggtccctcagactctcctg tgcag cctctgggttctccgtcagtggcacctacatgggctgggtccgccagg ctccagggaaggg actggagtgggtcgcacttctttatagtggtgg cgg cacataccacccag cgtccctg caggg ccg attcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaagccgag gacacg gccgtctattactgtgcg aaagg ag ggg cag gag gtg gccactttg actcctgg gg cca agg caccctggtgaccgtctcctca (SEQ ID NO. 105) Full QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK VL LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTKLTVLG (SEQ ID NO. 106) DNA cagtctgtgttgacgcagccg ccctcagtgtctgggg ccccagggcagagggtcaccatctcctgc actgggagcag ctccaacatcgggg caggttatgatgtacactggtaccag cagcttccaggaac agcccccaaactcctcatctatggtaacag caatcgg ccctcaggggtccctgaccgattctctggc tccaagtctggcacctcag cctccctgg ccatcagtgggctccagtctgaggatgagg ctgattatta ctgtg cagcatgggatgacag cctgaatggttatgtcttcggaactgggaccaagctgaccgtccta ggt (SEQ ID NO. 107) scFv QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFG TGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVETGGGLLQPG GSLRLSCAASGFSVSGTYMGWVRQAPGKGLEWVALLYSGGGTYHPA SLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVYYCAKGGAGGGHFDS WGQGTLVTVSS (SEQ ID NO. 108) WO 2014/143835 PCT/US2014/027977 -24 WT-1 (Ext002 #23) Antigen Peptide RMFPNAPYL (SEQ ID NO. 1) DNA cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta ggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag atggccgaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcaga ctctcctgtgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagg gaagggactggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgca gggccgattcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaa gccg ag gacacg g ccgtctattactgtgcg aaagg ag ggg cag gag gtg gccactttg actcctg gggccaaggcaccctggtgaccgtctcctca (SEQ ID NO. 109) Table 7 Antigen WT-1 (Ext002B #17) Peptide CMTWNQMNL (SEQ ID NO: 239) CDRs: 1 2 3 VH GYTLTELSMH GFDPEDGETIYAQKFQG SFYSYYGIDT (SEQ ID NO: 240) (SEQ ID NO: 241) (SEQ ID NO: 242) DNA ggatacaccctcactgaat ggttttgatcctgaagatggtgaaa tctttctactcttactacggta tatccatgcac caatctacgcacagaagttccagg tcgatact gc (SEQ ID NO: 243) (SEQ ID NO: 244) (SEQ ID NO: 245) VL QGDSLRRYYAS ANNNRPS NSRDISVNGWM (SEQ ID NO: 246) (SEQ ID NO: 247) (SEQ ID NO: 248) DNA caaggagacagcctcag gctaataacaatcggccctca aactcccgggacatcagt aaggtattatgcaagc gttaacggttggatg (SEQ ID NO: 249) (SEQ ID NO: 250) (SEQ ID NO: 251) Full QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLE VH WMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAV YYCARSFYSYYGIDTWGQGTLVTVSS (SEQ ID NO: 252) WO 2014/143835 PCT/US2014/027977 -25 Antigen WT-1 (Ext002B #17) Peptide CMTWNQMNL (SEQ ID NO: 239) DNA caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctg caaggtttccggatacaccctcactgaattatccatgcactgggtgcggcaggctcctggaaaagg gcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccaggg cagagtcaccatgaccg agg acacatctacagacacagcctacatg gag ctgag cagcctgag atctgaggacacggccgtgtattactgtgcgcgctctttctactcttactacggtatcgatacttggggtc aaggtactctggtgaccgtctcctca (SEQ ID NO: 253) Full SSELTQDPAVSVALGQTVRITCQGDSLRRYYASWYQQKPGQAPVLVI VL YANNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDISVN GWMFGGGTKLTVLG (SEQ ID NO: 254) DNA tcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgcca aggagacagcctcagaaggtattatgcaagctggtaccagcagaagccaggacaggcccctgt acttgtcatctatgctaataacaatcggccctcagggatcccagaccgattctctggctccagotcag gaaacacagcttccttgaccatcactggggctcaggcggaggatgaggctgactattattgtaactc ccgggacatcagtgttaacggttggatgttcggcggagggaccaagctgaccgtcctaggt (SEQ ID NO: 255) scFv SSELTQDPAVSVALGQTVRITCQGDSLRRYYASWYQQKPGQAPVLVI YANNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDISVN GWMFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGA EVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPED GETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARSFYSY YGIDTWGQGTLVTVSS (SEQ ID NO: 256) DNA tcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgcca aggagacagcctcagaaggtattatgcaagctggtaccagcagaagccaggacaggcccctgt acttgtcatctatgctaataacaatcggccctcagggatcccagaccgattctctggctccagotcag gaaacacagcttccttgaccatcactggggctcaggcggaggatgaggctgactattattgtaactc ccgggacatcagtgttaacggttggatgttcggcggagggaccaagctgaccgtcctaggttctag aggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggccc aggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgc aaggtttccggatacaccctcactgaattatccatgcactgggtgcggcaggctcctggaaaaggg cttgagtgg atg g gag gttttgatcctg aagatg gtg aaacaatctacg cacagaagttccag ggc agagtcaccatgaccg agg acacatctacagacacagcctacatg gag ctgag cagcctgag at ctgaggacacggccgtgtattactgtgcgcgctctttctactcttactacggtatcgatacttggggtca aggtactctggtgaccgtctcctca (SEQ ID NO: 257) WO 2014/143835 PCT/US2014/027977 -26 Table 8 Antigen WT-1 (Ext002B #28) Peptide CMTWNQMNL (SEQ ID NO: 239) CDRs: 1 2 3 VH GYTFTTYGMN WINTNTGKPTYAQGFTG GYYGWDYHDY (SEQ ID NO: 258) (SEQ ID NO: 259) (SEQ ID NO: 260) DNA ggatacaccttcactacct tggatcaacaccaacactgggaa ggttactacggttgggacta atggtatgaat gccaacgtatgcccagggttcac ccatgattac agga (SEQ ID NO: 261) (SEQ ID NO: 262) (SEQ ID NO: 263) VL GGNNIGSKSVH YDSDRPS QVWDSSSDHSPYV (SEQ ID NO: 264) (SEQ ID NO: 265) (SEQ ID NO: 266) DNA gggggaaacaacattgg tatgatagcgaccggccctca caggtgtgggatagtagta aagtaaaagtgtgcac gtgatcattccccttatgtc (SEQ ID NO: 267) (SEQ ID NO: 268) (SEQ ID NO: 269) Full QVQLVQSGSELKKPGASVKVSCKASGYTFTTYGMNWVRQAPGQGLE VH WMGWINTNTGKPTYAQGFTGRFVFSLDASVSTAYLQISGLKADDTAV YYCARGYYGWDYHDYWGQGTLVTVSS (SEQ ID NO: 270) DNA caggtgcagctggtgcagtctgggtctgagttgaagaagcctggggcctcagtgaaggtttcctgca aggcttctggatacaccttcactacctatggtatgaattgggtgcgacaggcccctggacaagggctt gagtggatgggatggatcaacaccaacactgggaagccaacgtatgcccagggcttcacagga cggtttgtcttctccttggacgcctctgtcagcacggcatatctgcagatcagcggcctaaaggctga cgacactgccgtgtattactgtgcgcgcggttactacggttgggactaccatgattactggggtcaag gtactctggtgaccgtctcctca (SEQ ID NO: 271) Full SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIY VL YDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD HSPYVFGTGTKLTVLG (SEQ ID NO: 272) DNA tcctatgtgctgactcagccaccctcagtgtcagtggccccaggaaagacggccaggattacctgt gggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccct gtgctggtcatctattatgatagcgaccggccctcagggatccctgagcgattctctggctccaactct gggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtc aggtgtgggatagtagtagtgatcattccccttatgtcttcggaactgggaccaagctgaccgtccta ggt (SEQ ID NO: 273) WO 2014/143835 PCT/US2014/027977 -27 Antigen WT-1 (Ext002B #28) Peptide CMTWNQMNL (SEQ ID NO: 239) scFv SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIY YDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD HSPYVFGTGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSG SELKKPGASVKVSCKASGYTFTTYGMNWVRQAPGQGLEWMGWINTN TGKPTYAQGFTGRFVFSLDASVSTAYLQISGLKADDTAVYYCARGYYG WDYHD (SEQ ID NO: 274) DNA tcctatgtgctgactcagccaccctcagtgtcagtggccccaggaaagacggccaggattacctgt gggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccct gtgctggtcatctattatgatagcgaccggccctcagggatccctgagcgattctctggctccaactct gggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtc aggtgtgggatagtagtagtgatcattccccttatgtcttcggaactgggaccaagctgaccgtccta ggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag atggcccaggtgcagctggtgcagtctgggtctgagttgaagaagcctggggcctcagtgaaggtt tcctgcaaggcttctggatacaccttcactacctatggtatgaattgggtgcgacaggcccctggaca agggcttgagtggatgggatggatcaacaccaacactgggaagccaacgtatgcccagggcttc acaggacggtttgtcttctccttggacgcctctgtcagcacggcatatctgcagatcagcggcctaaa ggctgacgacactgccgtgtattactgtgcgcgcggttactacggttgggactaccatgattactggg gtcaaggtactctggtgaccgtctcctca (SEQ ID NO: 275) WO 2014/143835 PCT/US2014/027977 -28 Table 9 Antigen WT-1 (Ext002B #39) Peptide CMTWNQMNL (SEQ ID NO: 239) CDRs: 1 2 3 VH GGTFSSYAIS GIIPIFGTANYAQKFQG WFYMQAGDH (SEQ ID NO: 276) (SEQ ID NO: 277) (SEQ ID NO: 278) DNA ggaggcaccttcagcagcta gggatcatccctatctttggtac tggttctacatgcaggctggt tgctatcagc agcaaactacgcacagaagtt gatcat ccagggc (SEQ ID NO: 279) (SEQ ID NO: 280) (SEQ ID NO: 281) VL TGSSSDVGTYNYDS DVSERPS SSFAASSPWL (SEQ ID NO: 282) (SEQ ID NO: 283) (SEQ ID NO: 284) DNA actggaagcagcagtgatgt gatgtcagtgagcggccctca agctcatttgcagccagcag tggtacttataactatgactct tccctggctg (SEQ ID NO: 285) (SEQ ID NO: 286) (SEQ ID NO: 287) Full QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE VH WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY CARWFYMQAGDHWGQGTLVTVSS (SEQ ID NO: 288) DNA caggtgcagctggtggagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg cagagtcacgattaccg cgg acg aatccacgag cacagcctacatg gag ctgag cagcctgag atctgaggacacggccgtgtattactgtgcgcgctggttctacatgcaggctggtgatcattggggtc aaggtactctggtgaccgtctcctca (SEQ ID NO: 289) Full QAVLTQPASVSGSPGQSITISCTGSSSDVGTYNYDSWYQQHPGKAPK VL LMIYDVSERPSGVSNRFSGSKSGNTAFLTISGLQAEDEADYYCSSFAA SSPWLFGGGTKVTVLG (SEQ ID NO: 290) DNA caggctgtgctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcact ggaagcagcagtgatgttggtacttataactatgactcttggtaccaacagcacccaggcaaagcc cccaaactcatgatttatgatgtcagtgagcggccctcaggggtttctaatcgcttctccggctccaag tctggcaacacggccttcctgaccatctctgggctccaggctgaggacgaggctgattattactgca gctcatttgcagccagcagtccctggctgttcggcggagggaccaaggtcaccgtcctaggt (SEQ ID NO: 291) WO 2014/143835 PCT/US2014/027977 -29 Antigen WT-1 (Ext002B #39) Peptide CMTWNQMNL (SEQ ID NO: 239) scFv QAVLTQPASVSGSPGQSITISCTGSSSDVGTYNYDSWYQQHPGKAPK LMIYDVSERPSGVSNRFSGSKSGNTAFLTISGLQAEDEADYYCSSFAA SSPWLFGGGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESG AEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFG TANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARWFYMQ AGDHWGQGTLVTVSS (SEQ ID NO: 292) DNA caggctgtgctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcact ggaagcagcagtgatgttggtacttataactatgactcttggtaccaacagcacccaggcaaagcc cccaaactcatgatttatgatgtcagtgagcggccctcaggggtttctaatcgcttctccggctccaag tctggcaacacggccttcctgaccatctctgggctccaggctgaggacgaggctgattattactgca gctcatttgcagccagcagtccctggctgttcggcggagggaccaaggtcaccgtcctaggttcta gaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcc caggtgcagctggtggagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg cagagtcacgattaccg cgg acg aatccacgag cacagcctacatg gag ctgag cagcctgag atctgaggacacggccgtgtattactgtgcgcgctggttctacatgcaggctggtgatcattggggtc aaggtactctggtgaccgtctcctca (SEQ ID NO: 293) Table 10 Antigen WT-1 (Ext002B #40) Peptide CMTWNQMNL (SEQ ID NO: 239) CDRs: 1 2 3 VH GFTFSSYGMN SISSSSSYIYYADSVKG IQDATGEEMILYDY (SEQ ID NO: 294) (SEQ ID NO: 295) (SEQ ID NO: 296) DNA ggattcaccttcagtagct tccattagtagtagtagtagttac atccaggacgctactggtgaa atggcatgaac atatactacgcagactcagtga gaaatgatcctgtacgattac agggc (SEQ ID NO: 297) (SEQ ID NO: 298) (SEQ ID NO: 299) VL RSSQSLVYSDGN QVSKRDS MQGSHLRT TYLN (SEQ ID NO: 300) (SEQ ID NO: 301) (SEQ ID NO: 302) WO 2014/143835 PCT/US2014/027977 -30 Antigen WT-1 (Ext002B #40) Peptide CMTWNQMNL (SEQ ID NO: 239) DNA aggtctagtcaaagcctc caggtttctaagcgggactct atgcaaggttcacacttgcgg gtatacagtgatggaaac acg acctatttgaat (SEQ ID NO: 303) (SEQ ID NO: 304) (SEQ ID NO: 305) Full QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMNWVRQAPGKGLE VH WVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCARIQDATGEEMILYDYWGQGTLVTVSS (SEQ ID NO: 306) DNA caggtgcagctggtggagtctgggggaggcctggtcaagcctgggggggtccctgaggctctcctgt gcagcctctggattcaccttcagtagctatggcatgaactgggtccgccaggctccagggaagggg ctggagtgggtctcatccattagtagtagtagtagttacatatactacgcagactcagtgaagggccg attcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccg aggacacggctgtgtattactgtgcgcgcatccaggacgctactggtgaagaaatgatcctgtacg attactggggtcaaggtactctggtgaccgtctcctca (SEQ ID NO: 307) Full EIVLTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQS VL PRRLIYQVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGMYYCMQ GSHLRTFGQGTKVEIKR (SEQ ID NO: 308) DNA attgtgctgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgcagg tctagtcaaagcctcgtatacagtgatggaaacacctatttgaattggtttcagcagaggccaggcc aatctccaaggcgcctaatttatcaggtttctaagcgggactctggggtcccagacagattcagcgg cagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttgggatgta ttactgcatgcaaggttcacacttgcggacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO: 309) scFv EIVLTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQS PRRLIYQVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGMYYCMQ GSHLRTFGQGTKVEIKRSRGGGGSGGGGSGGGGSLEMAQVQLVES GGGLVKPGGSLRLSCAASGFTFSSYGMNWVRQAPGKGLEWVSSISS SSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIQDA TGEEMILYDYWGQGTLVTVSS (SEQ ID NO: 310) WO 2014/143835 PCT/US2014/027977 -31 Antigen WT-1 (Ext002B #40) Peptide CMTWNQMNL (SEQ ID NO: 239) DNA gaaattgtgctgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgc aggtctagtcaaagcctcgtatacagtgatggaaacacctatttgaattggtttcagcagaggccag gccaatctccaaggcgcctaatttatcaggtttctaagcgggactctggggtcccagacagattcag cgg cagtg ggtcag gcactg atttcacactgaaaatcagcagg gtg gag g ctgag g atgttgg ga tgtattactgcatgcaaggttcacacttgcggacgttcggccaagggaccaaggtggaaatcaaac gttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagat ggcccaggtgcagctggtggagtctgggggaggcctggtcaagcctgggggggtccctgaggctct cctgtgcagcctctggattcaccttcagtagctatggcatgaactgggtccgccaggctccagggaa ggggctggagtgggtctcatccattagtagtagtagtagttacatatactacgcagactcagtgaag ggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgag agccgaggacacggctgtgtattactgtgcgcgcatccaggacgctactggtgaagaaatgatcct gtacgattactggggtcaaggtactctggtgaccgtctcctca (SEQ ID NO: 311) Table 11 Antigen WT-1 (Ext002B #41) Peptide CMTWNQMNL (SEQ ID NO: 239) CDRs: 1 2 3 VH GYTFTDYYIH WMNPNSGNSVSAQKFQG YQGSTWKYDSYGDL (SEQ ID NO: 312) (SEQ ID NO: 313) (SEQ ID NO: 314) DNA ggatacaccttcaccg tggatgaaccctaacagtgggaactca taccagggttctacttggaaa actactatatacac gtctctgcacagaagttccagggc tacgactcttacggtgatctg (SEQ ID NO: 315) (SEQ ID NO: 316) (SEQ ID NO: 317) VL GGNEIGFNGVH NNRVRPS QVWVNPDNEYV (SEQ ID NO: 318) (SEQ ID NO: 319) (SEQ ID NO: 320) DNA gggggaaacgagatt aacaatagggtccggccctca caggtgtgggttaatcctgat ggatttaatggtgttcat aatgaatatgtc (SEQ ID NO: 321) (SEQ ID NO: 322) (SEQ ID NO: 323) Full QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWM VH GWMNPNSGNSVSAQKFQGRVTMTRDTSINTAYMELSSLTSDDTAVYYC ARYQGSTWKYDSYGDLWGQGTLVTVTS (SEQ ID NO: 324) WO 2014/143835 PCT/US2014/027977 -32 Antigen WT-1 (Ext002B #41) Peptide CMTWNQMNL (SEQ ID NO: 239) DNA caggtccagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgca aggcttctggatacaccttcaccgactactatatacactgggtgcggcaggcccctggacaagggctg gagtggatgggatggatgaaccctaacagtgggaactcagtctctgcacagaagttccagggcagag tcaccatgaccagggatacctccataaacacagcctacatggagctgagcagcctgacatctgacga cacggccgtatattactgtgcgcgctaccagggttctacttggaaatacgactcttacggtgatctgtggg gtcaaggtactctggtgaccgtcacctca (SEQ ID NO: 325) Full QAVLTQPPSVSVAPGETATVTCGGNEIGFNGVHWYKQKAGQAPLLVIYN VL NRVRPSGISERLSGSNSGNTATLTISRVEAGDEADYYCQVWVNPDNEYV FGSGTKVTVLG (SEQ ID NO: 326) DNA cagg ctgtgctg actcagccaccctcg gtgtcagtgg ccccag gag ag acg gccactgttacctgtg g gggaaacgagattggatttaatggtgttcattggtataagcagaaggcaggccaggcccctctgttggtc atctataacaatagggtccggccctcagggatctctgagcgactctctggctccaactctggtaacacgg ccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggttaat cctgataatgaatatgtcttcggatcggggaccaaggtcaccgtcctaggt (SEQ ID NO: 327) scF QAVLTQPPSVSVAPGETATVTCGGNEIGFNGVHWYKQKAGQAPLLVIYN v NRVRPSGISERLSGSNSGNTATLTISRVEAGDEADYYCQVWVNPDNEYV FGSGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKP GASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWMNPNSGNSVSA QKFQGRVTMTRDTSINTAYMELSSLTSDDTAVYYCARYQGSTWKYDSY GDLWGQGTLVTVTS (SEQ ID NO: 328) DNA cagg ctgtgctg actcagccaccctcg gtgtcagtgg ccccag gag ag acg gccactgttacctgtg g gggaaacgagattggatttaatggtgttcattggtataagcagaaggcaggccaggcccctctgttggtc atctataacaatagggtccggccctcagggatctctgagcgactctctggctccaactctggtaacacgg ccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggttaat cctgataatgaatatgtcttcggatcggggaccaaggtcaccgtcctaggttctagaggtggtggtggt agcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtccagctggtgcag tctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttc accgactactatatacactgggtgcggcaggcccctggacaagggctggagtggatgggatggatga accctaacagtgggaactcagtctctgcacagaagttccagggcagagtcaccatgaccagggatac ctccataaacacagcctacatggagctgagcagcctgacatctgacgacacggccgtatattactgtgc gcgctaccagggttctacttggaaatacgactcttacggtgatctgtggggtcaaggtactctggtgacc gtcacctca (SEQ ID NO: 329) WO 2014/143835 PCT/US2014/027977 -33 Table 12 Antigen WT-1 (Ext002B #43) Peptide CMTWNQMNL (SEQ ID NO: 239) CDRs: 1 2 3 VH GFTFSSYEMN YISSSGSTIYYADSVKG DWRSSYYYSQYDK (SEQ ID NO: 330) (SEQ ID NO: 331) (SEQ ID NO: 332) DNA ggattcaccttcagtagtta tacattagtagtagtggtagtacc gactggcgttcttcttactacta tgaaatgaac atatactacgcagactctgtgaa ctctcagtacgataaa 9990 (SEQ ID NO: 333) (SEQ ID NO: 334) (SEQ ID NO: 335) VL TRSSGNIASNYVQ ADNQRPS QSYENNIHV (SEQ ID NO: 336) (SEQ ID NO: 337) (SEQ ID NO: 338) DNA acccgcagcagtggcaa gcggacaaccaaagaccctct cagtcttatgaaaacaacatt cattgccagcaactatgtg cacgtg cag (SEQ ID NO: 339) (SEQ ID NO: 340) (SEQ ID NO: 341) Full EVQLVESGGGLVQPGESLRLSCAASGFTFSSYEMNWVRQAPGKGLE VH WVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCARDWRSSYYYSQYDKWGQGTLVTVSS (SEQ ID NO: 342) DNA gaggtgcagctggtggagtctgggggaggcttggtacagcctggagagtccctgagactctcctgt gcagcctctggattcaccttcagtagttatgaaatgaactgggttcgccaggctccagggaagggg ctggagtgggtttcatacattagtagtagtggtagtaccatatactacgcagactctgtgaagggccg attcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccg aggacacggctgtgtattactgtgcgcgcgactggcgttcttcttactactactctcagtacgataaatg gggtcaaggtactctggtgaccgtctcctca (SEQ ID NO: 343) Full NFMLTQPHSVSESPGKTVSISCTRSSGNIASNYVQWYQHRPGRSPTT VL VIYADNQRPSGVPDRFSGSIDTSSNSASLTISGLRTEDEADYYCQSYE NNIHVFGGGTKLTVLG (SEQ ID NO: 344) DNA aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaagcatctcctgcac ccgcagcagtggcaacattgccagcaactatgtgcagtggtaccaacaccgcccgggccgttccc ccaccactgtgatctatgcggacaaccaaagaccctctggggtccctgatcgcttctctggctccatc gacacctcctccaactctgcctccctcaccatctctggactgaggactgaggacgaggctgactact actgtcagtcttatgaaaacaacattcacgtgttcg0ggggggaccaagctgaccgtcctaggt (SEQ ID NO: 345) WO 2014/143835 PCT/US2014/027977 -34 Antigen WT-1 (Ext002B #43) Peptide CMTWNQMNL (SEQ ID NO: 239) scFv NFMLTQPHSVSESPGKTVSISCTRSSGNIASNYVQWYQHRPGRSPTT VIYADNQRPSGVPDRFSGSIDTSSNSASLTISGLRTEDEADYYCQSYE NNIHVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAEVQLVESG GGLVQPGESLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSS GSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWRS SYYYSQYDKWGQGTLVTVSS (SEQ ID NO: 346) DNA aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaagcatctcctgcac ccgcagcagtggcaacattgccagcaactatgtgcagtggtaccaacaccgcccgggccgttccc ccaccactgtgatctatgcggacaaccaaagaccctctggggtccctgatcgcttctctggctccatc gacacctcctccaactctgcctccctcaccatctctggactgaggactgaggacgaggctgactact actgtcagtcttatgaaaacaacattcacgtgttcggcggggggaccaagctgaccgtcctaggttc tagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggc cgaggtgcagctggtggagtctgggggaggcttggtacagcctggagagtccctgagactctcctg tgcagcctctggattcaccttcagtagttatgaaatgaactgggttcgccaggctccagggaagggg ctggagtgggtttcatacattagtagtagtggtagtaccatatactacgcagactctgtgaagggccg attcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccg aggacacggctgtgtattactgtgcgcgcgactggcgttcttcttactactactctcagtacgataaatg gggtcaaggtactctggtgaccgtctcctca (SEQ ID NO: 347) [0089] In the sequences in Tables 1-14, bolded text indicates a linker sequence between hypervariable heavy and light chain sequences. [0090] In some embodiments, anti-WT-1 antibodies used in the method of the invention may further encompass those comprising light and heavy hypervariable regions and constant regions, for example as shown in Tables 13 (heavy chain), 14 (light chain) and 15 (constant regions). Similarly, the CDRs of other WT-1 antibodies suitable for use in practicing the disclosed method are shown in Figures 7-10.
WO 2014/143835 PCT/US2014/027977 -35 Table 13 CDR-H1 CDR-H2 CDR-H3 SEQ ID NO. roup I XT002-12(166) SNAVAWN RTYRGSTYY---ALSV G-SNSAFDF 119-121 XT002-5(184) SNSAAWN RTYYGSKWYNDYAVSV GRLGDAFDI 122-124 XT002-8(184) SDGAAWN RTYYRSKWYNDYAVSV GDYYYGMDV 125-127 onsensus(191) SNAAAWN RTYYGSKWYNDYAVSV G AFDI 128-130 roup II XT002-14(163) SYWIS RIDPSDSYTNYSPSFQG GD------YDFYLDP-- 131-133 XT002-25(163) SYGIS WISAYNGNTNYAQKLQG DLYSSGWYESYYYGMDV 134-136 XT002-3(186) SYAIS GIIPIFGTANYAQKFQG RIP-P------YYGMDV 137-139 XT002-30(163) SYGIS WISAHNGNTNYAQKLQG DR-------VWFGDLSD 134, 140 141 XT002-33(163) SYAIS GIIPIFGTANYAQKEQG NYDFWSG-----DAFDI 137, 142 143 onsensus(188) SYAIS I P G TNYAQKFQG FY GMDV 137, 144 145 roup III XT002-34(161) DYGMS GINWNGGSTGYADSV ERGY-GYHDPHDY 146-148 XT002-40(157) NYTMN SISLSGAYIYYADSL EGYSSSVYDAFDL 149-151 XT002-45(165) SYGMH GILSDGGKDYYVDSV CSSN-YGNDAFDI 152-154 XT002-48(165) TYSMN SISSGAYSIFYADSV DQYYGDKWDAFDI 155-157 onsensus (170) SYGMN SISS GGSIYYADSV E YY WDAFDI 158-160 WO 2014/143835 PCT/US2014/027977 -36 Table 14 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, WO 2014/143835 PCT/US2014/027977 -37 CDR-L1 CDR-L2 CDR-L3 SEQ ID NOS. Group I 162, 164 Consensus (46) CSGSSSNIGS N V NNQRPSG AAWDDSL G WVFG 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 QQSYSTP LT 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 WO 2014/143835 PCT/US2014/027977 -38 CDR-L1 CDR-L2 CDR-L3 SEQ ID NOS. Group I 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 SSYTNTGTV--L 230-232 EXT002-4 (47) TGTSSDFGDYDYVS DVSDRPS QSYDSSLSGSGV 233-235 Consensus (47) TGTSSDVGGYNYVS DVS RPS SSYTSS S V 225, 234, 229 WO 2014/143835 PCT/US2014/027977 -39 Table 15 Constant Regions Human heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL constant region and TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT IgGI Fcdomain 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) WO 2014/143835 PCT/US2014/027977 -40 [0091] In some embodiments, the anti-WT-1 antibodies are those in which 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). [0092] In one embodiment, the antibody is an anti -WT-1/A2 antibody and comprises the human IgG1 constant region and Fc domain shown in Table 9. In one embodiment, the anti-WT-1/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 1-14 and in Figures 7-10. [0093] Glycosylation (specifically fucosylation ) variants of IgG Fc can be produced using host expression cells and methods described in U.S. patents 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. [0094] According to an aspect of some embodiments of the disclosure there is provided a method of treating cancer in a subject in need thereof. The method, according to these embodiments, is effected by administering to the subject a WO 2014/143835 PCT/US2014/027977 -41 therapeutically effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an anti-WT-1 antibody. [0095] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition, and includes, for example, reducing a size of a tumor in a subject, effecting a state of remission in a subject, increasing an expected survival probability, increasing life expectancy, and increasing an expected time to disease progression. [0096] As described in the Examples section that follows, tyrosine kinase inhibitors such as imatinib and dasatinib and anti-WT-1 antibodies were surprisingly observed to have a beneficial additive effect when administered together. Importantly, several animals administered the combination of dasatinib and anti-WT-1 antibody appeared to be cured of their disease whereas animals administered either drug alone were not. [0097] Suitable routes of administration for the TKI may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. [0098] Oral administration is an exemplary administration for tyrosine kinase inhibitors. It is to be understood that administration of a tyrosine kinase inhibitor and anti-WT-1 antibody need not be via the same route, and need not be performed simultaneously. [0099] WT-1 (or anti-WT-1) antibodies will vary in the nature of the antigen to which they bind. Specificity is determined by HLA antigen type. For example, HLA-A*0201 is expressed in 39-46% of all Caucasians and therefore, an antibody with specificity for WT-1 peptide in conjunction with HLA-A2 represents a suitable choice of antibody WO 2014/143835 PCT/US2014/027977 -42 for use in the Caucasian population. Anti-WT-1 antibodies with specificity for a WT-1 peptide presented on the surface of cancer cells in conjunction with HLA-A24 may be more appropriate for use in New World natives and Asian populations in which the HLA-A24 target is particularly expressed. Choice of WT-1 antibody, therefore, may depend on HLA type of the subject to whom it is to be administered. [00100] In other embodiments, the anti-WT-1/HLA 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. [00101] Methods for reducing the proliferation of leukemia cells is also included, comprising contacting leukemia cells with a WT-1 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. Pharmaceutical Compositions and Methods of Treatment [00102] WT-1 antibodies can be administered for therapeutic treatments to a patient suffering from a tumor or WT-1 -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. [00103] The identification of medical conditions treatable by WT-1 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 WT-1 antibodies. A clinician skilled in the WO 2014/143835 PCT/US2014/027977 -43 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. [00104] Non-limiting examples of pathological conditions characterized by WT-1 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 WT-1 antibodies. [00105] Any suitable method or route can be used to administer a WT-1 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. [00106] It is understood that WT-1 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. [00107] Other aspects of the invention include without limitation, the use of antibodies and nucleic acids that encode them for treatment of WT-1 associated disease, for diagnostic and prognostic applications as well as use as research tools for the detection of WT-1 in cells and tissues. Pharmaceutical compositions comprising the disclosed antibodies and nucleic acids are encompassed by the WO 2014/143835 PCT/US2014/027977 -44 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. [00108] Embodiments contemplated in view of the foregoing description include, but are not limited to, the following numbered embodiments: [00109] 1. A pharmaceutical composition comprising a tyrosine kinase inhibitor (TKI); and (A) an antibody comprising a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, comprising amino acid sequences shown in Tables 1-14 and Figures 7-10; or (B) an antibody comprising VH and VL comprising first and second amino acid sequences from Tables 1-12; or (C) an antibody comprising an scFv comprising an amino acid sequence from Tables 1-12. [00110] 2. The pharmaceutical composition of embodiment 1, wherein the tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib. [00111] 3. The pharmaceutical composition of embodiment 1, wherein the tyrosine kinase inhibitor is imatinib, ponatinib or dasatinib or a pharmaceutically acceptable salt thereof. [00112] 4. The pharmaceutical composition of embodiment 1, wherein the pharmaceutically acceptable salt of imatinib is imatinib mesylate.
WO 2014/143835 PCT/US2014/027977 -45 [00113] 5. The pharmaceutical composition of embodiment 1, wherein the pharmaceutically acceptable salt of ponatinib is ponatinib hydrochloride. [00114] 6. A method for treating or inhibiting the proliferation of a WT-1 positive cancer, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an anti-WT-1 antibody or antigen-binding fragment thereof. [00115] 7. The method of embodiment 6, wherein said WT-1 positive cancer is selected from the group consisting of chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS), gastrointestinal stromal tumor, ovarian cancer, prostate cancer, soft tissue sarcoma, and malignant glioma. [00116] 8. The method of embodiment 6, wherein the tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib. [00117] 9. The method of embodiment 6, wherein the tyrosine kinase inhibitor is imatinib, ponatinib or dasatinib or a pharmaceutically acceptable salt thereof. [00118] 10. The method of embodiment 6, wherein the pharmaceutically acceptable salt of imatinib is imatinib mesylate. [00119] 11. The method of embodiment 6, wherein the pharmaceutically acceptable salt of ponatinib is ponatinib hydrochloride. [00120] 12. The method of embodiment 6, wherein said anti-WT-1 antibody is selected from the group consisting of: (A) an antibody comprising a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, comprising amino acid sequences shown in Tables 1-14 and Figures 7-10; or WO 2014/143835 PCT/US2014/027977 -46 (B) an antibody comprising VH and VL comprising first and second amino acid sequences from Tables 1-12; or (C) an antibody comprising an scFv comprising an amino acid sequence from Tables 1-12. [00121] 13. The method of embodiment 6, wherein the anti-WT-1 antibody comprises a human variable region framework region. [00122] 14. The method of embodiment 6, wherein the anti-WT-1 antibody is fully human. [00123] 15. The method of embodiment 6, wherein the anti-WT-1 antibody, or antigen-binding portion thereof, specifically binds a WT-1 peptide in an HLA restricted manner. [00124] 16. The method of embodiment 6, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof, binds to WT-1/HLA with a K 0 of 1X1 08 M or less. [00125] 17. The method of embodiment 6, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof, binds to WT-1/HLA with a K 0 of about 1X10 11 M to about 1X10- 8 M. [00126] 18. The method of embodiment 6, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof, induces antibody dependent cellular cytotoxicity (ADCC) against WT-1 -positive cells. [00127] 19. The method of embodiment 6, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof inhibits growth of WT-1 positive cells in vivo. [00128] 20. The method of embodiment 6, wherein the antigen-binding fragment of said antibody is an Fab, Fab', F(ab') 2 , Fv or single chain Fv (scFv). [00129] The method of the present invention will now be described in more detail with respect to representative embodiments. EXAMPLE 1 WO 2014/143835 PCT/US2014/027977 -47 Materials and Methods [00130] 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. All cells were HLA typed by the Department of Cellular Immunology at Memorial Sloan-Kettering Cancer Center. Leukemia cell line, BV1 73, (WT-1 +, A0201 +) was kindly provided by Dr. H. J. Stauss (University College London, London, United Kingdom). The cell lines were cultured in RPMI 1640 supplemented with 5% FCS, penicillin, streptomycin, 2 mmol/L glutamine, and 2-mercaptoethanol at 370 C/5% CO 2 . [00131] Animals. Six to eight week-old male NOD.Cg-Prkdc scid IL2rgtmlWjl/SzJ mice, known as NOD/SCID gamma (NSG), were purchased from the Jackson Laboratory (Bar Harbor, ME) or obtained from MSKCC animal breeding facility. [00132] Transduction and selection of luciferase/GFP positive cells. BV1 73 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. EXAMPLE 2 Antibody-dependent cellular cytotoxicity (ADCC) [00133] ADCC is considered to be one of the major effector mechanisms of therapeutic mAb in humans. Evaluation of efficacy, therefore, begins with in vitro experiments measuring ADCC against BV1 73 cell line, derived from CML in blastic crisis. Fresh BV1 73 cells were used for ADCC target cells. WT-1 antibody or its isotype control human IgG1 was incubated at 750 ng/ml with target cells and fresh PBMCs at different effector: target (E:T) ratio for 6 hrs. Imatinib was added at concentrations of 0, 1, 5, and 10 pM. The supernatants were harvested and the cytotoxicity was measured by standard chromium 51 release assay.
WO 2014/143835 PCT/US2014/027977 -48 [00134] In the presence of human PBMC, WT-1 antibody mediated dose-dependent PBMC ADCC against naturally presented RMF epitope by HLA-A0201 molecule on tumor cells, the leukemia cell line BV1 73. Importantly, WT-1 antibody was able to mediate ADCC in the presence of various doses of imatinib. The killing was consistently observed at 750 ng/ml of WT-1 antibody using PBMCs as effector cells from multiple healthy donors. These results demonstrated that imatinib does not affect the ability of WT-1 antibody to mediate specific ADCC against cells that naturally express RMF and HLA-A0201 complex in vitro (Figure 1). EXAMPLE 3 [00135] In vivo efficacy of ESKM with TKIs was evaluated using NSG mice injected with HLA-A0201* leukemic cell line BV173. The protocol used for imatinib and dasatinib therapy in combination with ESKM consisted of injecting 3 x 106 cells per mouse via tail vein, luciferin imaging 6 days after injection to assess tumor engraftment, and initiation of therapy immediately after imaging on day 6. Luciferin imaging was used weekly to monitor tumor growth. The TKI is injected intraperitoneally daily (50mg/kg for imatinib and 20-40mg/kg for dasatinib.) The antibody is injected intravenously twice per week. EXAMPLE 4 [00136] Therapeutic effects of imatinib plus anti- WT- 1/HLA antibody (ESKM) in a human leukemia xenograft NSG model. Three million BV1 73 human leukemia cells were injected IV by tail vein into NSG mice. On day 6, 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 (A, B, C, and D). Immediately after imaging on day 6, therapy was initiated with anti- WT-1 antibody ESKM 100 pg administered by intraperitoneal (IP) injection twice weekly. Imatinib was also administered by IP injection at 50 mg/kg daily. Therapy continued for 5 weeks (10 doses of ESKM and 34 doses of imatinib per mouse). Group A: No therapy; Group B: imatinib treatment only; Group C: ESK treatment only; Group D: combination of both imatinib daily and ESK twice weekly. Tumor growth was assessed by luminescence imaging weekly, and clinical activity was assessed daily.
WO 2014/143835 PCT/US2014/027977 -49 [00137] After 5 weeks of therapy, animals were imaged by fluorescent luciferin imaging, and the fluorescence was quantified using Living Image@ software. This allows for the quantification of mouse tumor burden. The results are shown in Figure 3. Animals that received only imatinib (50 mg/kg daily) had reduced tumor burden compared to control animals that received neither imatinib nor anti-WT-1 antibody. Animals that received 100 pg of anti-WT-1 antibody twice a week for 5 weeks were much improved over control mice and imatinib-treated mice. The largest reduction of tumor cells was found in animals that received the combination of anti-WT-1 antibody and imatinib (Group D). These animals also showed reduced growth of tumor, evident from their previous day of imaging a week earlier. (Figure 2) EXAMPLE 5 [00138] Therapeutic effects of dasatinib plus anti-WT- 1/HLA antibody (ESKM) in a human leukemia xenograft NSG model. Three million BV1 73 human leukemia cells were injected IV by tail vein into NSG mice. On day 6, tumor engraftment was confirmed by firefly luciferase imaging in all mice that were to be treated; mice were then randomly divided into five different treatment groups (A, B, C, D, and E). Immediately after imaging on day 6, therapy was initiated with anti-WT-1 antibody ESKM 100 pg administered by intraperitoneal (IP) injection twice weekly. Dasatinib was also administered by IP injection at 40 mg/kg daily. Since dasatinib is not soluble in aqueous solution, it was administered dissolved in 50 PL DMSO. Group A: No therapy; Group B: DMSO only (vehicle control); Group C: dasatinib treatment only; Group D: ESK treatment only; Group E: combination of both dasatinib daily and ESK twice weekly. After 7 days of therapy, it was noted that the mice treated with dasatinib looked ill with significant weight loss. The dose was decreased to 20 mg/kg. The mice continued to be in poor health, with 1 death, and on day 11 of therapy dasatinib was discontinued due to toxicity. ESK antibody continued to be administered for the full 4 week treatment cycle. Tumor growth continued to be assessed by luminescence imaging weekly. [00139] After 4 weeks of therapy, animals were imaged by fluorescent luciferin imaging, and the fluorescence was quantified using Living Image@ software, quantifying mouse tumor burden. The results are shown in Figure 6. Animals that WO 2014/143835 PCT/US2014/027977 -50 received only dasatinib initially had clearance of tumor, but relapsed by week 4 of therapy. Animals that received 100 pg of anti-WT-1 antibody twice a week for 4 weeks were much improved over control mice, though they had increased tumor burden compared to the dasatinib only mice. Of the mice that received combination of dasatinib and ESK, one mouse had intracranial tumor relapse, and three others remain tumor free. The fifth mouse died from dasatinib toxicity on day 8 of therapy. EXAMPLE 6 [00140] BV1 73 is an HLA-A0201 +, human Ph+ ALL cell line that expresses WT1, and is tagged with luciferase. A tyrosine kinase inhibitor (TKI) resistant BV173-R cell line was engineered by transducing BV1 73 with the resistant T3151 Bcr-Abl plasmid. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. Tumor growth in vivo was assessed in NOD/SCID gamma (NSG) mice with bioluminescence imaging (BLI). RT-PCR was used to evaluate minimal residual disease in mice with negative BLI signal at the end of therapy. Imatinib, dasatinib, and ponatinib were used at up to maximally tolerated doses, given IP once daily. ESKM was administered at 100 pg twice weekly IP. [00141] ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro. In a BV1 73 engrafted human leukemia xenograft model, ESKM was more potent than imatinib, with median tumor growth reduction of 78% vs 52%. Combination of imatinib and ESKM therapy resulted in a 94% reduction in leukemic growth. High dose dasatinib (40 mg/kg daily) was more potent than ESKM, but discontinuation of therapy due to dasatinib toxicity resulted in relapse. Combination with ESKM therapy with dasatinib resulted in cure in 75% of mice, confirmed by bone marrow RT-PCR three weeks after termination of therapy. For mice cytoreduced with dasatinib followed by consolidation therapy with ESKM, delayed relapse was observed, but no cures. ESKM was highly superior to imatinib and dasatinib against the T3151 BV1 73 R leukemia in vivo. Cures were not achieved with combination therapy of ESKM and either first or second generation TKIs against resistant T3151 leukemia. [00142] Ponatinib at 10 mg/kg had higher efficacy than ESKM alone against BV173 R, but mice treated with combination of ESKM and ponatinib had superior tumor WO 2014/143835 PCT/US2014/027977 -51 reduction (Figure 13). The conclusion is that ESKM is an effective therapeutic antibody for sensitive and T3151 Ph+ ALL. Resistant T3151 Ph+ leukemic growth is inhibited more effectively by ESKM therapy compared to imatinib and dasatinib, and combination therapy with ESKM is superior to ponatinib.
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Claims (21)

1. A pharmaceutical composition comprising a tyrosine kinase inhibitor (TKI); and (A) an antibody comprising a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, comprising amino acid sequences shown in Tables 1-14 and Figures 7-10; or (B) an antibody comprising VH and VL comprising first and second amino acid sequences from Tables 1-12; or (C) an antibody comprising an scFv comprising an amino acid sequence from Tables 1-12.
2. The pharmaceutical composition of claim 1, wherein the tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib.
3. The pharmaceutical composition of claim 1, wherein the tyrosine kinase inhibitor is imatinib, ponatinib or dasatinib or a pharmaceutically acceptable salt thereof.
4. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt of imatinib is imatinib mesylate.
5. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt of ponatinib is ponatinib hydrochloride.
6. A tyrosine kinase inhibitor and an anti-WT-1 antibody or antigen-binding fragment thereof for use in treating or inhibiting the proliferation of a WT-1 positive cancer.
7. A method for treating or inhibiting the proliferation of a WT-1 positive cancer, the method comprising administering to a subject in need thereof, a therapeutically WO 2014/143835 PCT/US2014/027977 -59 effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an anti-WT-1 antibody or antigen-binding fragment thereof.
8. The use or method of claim 6 or 7, wherein said WT-1 positive cancer is selected from the group consisting of chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS), gastrointestinal stromal tumor, ovarian cancer, prostate cancer, soft tissue sarcoma, and malignant glioma.
9. The use or method of claim 6 or 7, wherein the tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib.
10. The use or method of claim 6 or 7, wherein the tyrosine kinase inhibitor is imatinib, ponatinib or dasatinib or a pharmaceutically acceptable salt thereof.
11. The use or method of claim 6 or 7, wherein the pharmaceutically acceptable salt of imatinib is imatinib mesylate.
12. The use or method of claim 6 or 7, wherein the pharmaceutically acceptable salt of ponatinib is ponatinib hydrochloride.
13. The use or method of claim 6 or 7, wherein said anti-WT-1 antibody is selected from the group consisting of: (A) a antibody comprising a heavy chain (HC) variable region comprising HC CDR1, HC-CDR2 and HC-CDR3; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, comprising amino acid sequences shown in Tables 1-14 and Figures 7-10; or (B) an antibody comprising VH and VL comprising first and second amino acid sequences from Tables 1-12; or (C) an antibody comprising an scFv comprising an amino acid sequence from Tables 1-12. WO 2014/143835 PCT/US2014/027977 -60
14. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody comprises a human variable region framework region.
15. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody is fully human.
16. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody, or antigen-binding portion thereof, specifically binds a WT-1 peptide in an HLA restricted manner.
17. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof, binds to WT-1/HLA with a K 0 of 1X1 08 M or less.
18. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof, binds to WT-1/HLA with a K 0 of about 1X1 0 11 M to about 1X10- 8 M.
19. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof, induces antibody dependent cellular cytotoxicity (ADCC) against WT-1 -positive cells.
20. The use or method of claim 6 or 7, wherein the anti-WT-1 antibody, or an antigen-binding portion thereof inhibits growth of WT-1 positive cells in vivo.
21. The use or method of claim 6 or 7, wherein the antigen-binding fragment of said antibody is an Fab, Fab', F(ab') 2 , Fv or single chain Fv (scFv).
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