EP2968539A1 - Combination/adjuvant therapy for wt-1-positive disease - Google Patents

Combination/adjuvant therapy for wt-1-positive disease

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Publication number
EP2968539A1
EP2968539A1 EP14722878.7A EP14722878A EP2968539A1 EP 2968539 A1 EP2968539 A1 EP 2968539A1 EP 14722878 A EP14722878 A EP 14722878A EP 2968539 A1 EP2968539 A1 EP 2968539A1
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
antigen
imatinib
tyrosine kinase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14722878.7A
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German (de)
French (fr)
Inventor
David Scheinberg
Leonid DUBROVSKY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Memorial Sloan Kettering Cancer Center
Original Assignee
Sloan Kettering Institute for Cancer Research
Memorial Sloan Kettering Cancer Center
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Application filed by Sloan Kettering Institute for Cancer Research, Memorial Sloan Kettering Cancer Center filed Critical Sloan Kettering Institute for Cancer Research
Publication of EP2968539A1 publication Critical patent/EP2968539A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3038—Kidney, bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • 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 inhibitor therapeutic agent and antibodies against Wilm's tumor oncogene protein (WT-1 ).
  • CML chronic myelogenous leukemia
  • WT-1 Wilm's tumor oncogene protein
  • Tyrosine kinase inhibitors 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
  • MDS myelodysplastic syndrome
  • ovarian cancer prostate cancer
  • soft tissue sarcoma malignant glioma
  • renal cell cancer hepatocellular carcinoma
  • gastrointestinal stromal tumor GIST
  • breast cancer breast cancer
  • lung cancer etc.
  • TKIs for example, imatinib and sunitinib
  • the clinical efficacy of some TKIs are limited by rare patient-specific intolerance to the drug or the development of treatment-refractory disease.
  • WT-1 Wilms' tumor oncogene protein
  • CML CML
  • 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).
  • 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).
  • antibodies to WT-1 are detected in patients with hematopoietic malignancies and solid tumors, indicating that WT-1 is a highly immunogenic antigen (7).
  • mAbs therapeutic monoclonal antibodies
  • WT-1 is a nuclear protein and, therefore, is inaccessible to classical antibody therapy.
  • 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.
  • CTL cytotoxic CD8 T cell
  • 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.
  • CDC complement- dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • direct cell inhibition or apoptosis-inducing effects on tumor cells that over-express the target molecules include 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.
  • 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 of antibodies that recognize and bind to peptides of WT-1 oncoprotein in an HLA- restricted fashion.
  • TKIs tyrosine kinase inhibitors
  • 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.
  • 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.
  • 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.
  • Bcr-Abl imatinib, dasatinib and nilotinib
  • EGFR erlotinib and gefitinib
  • VEGFR-1 pazopanib and sorafenib
  • 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.
  • CML chronic myelogenous leukemia
  • MM multiple myeloma
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid/myelogenous leukemia
  • MDS myelodysplastic syndrome
  • mesothelioma mesothelioma
  • ovarian cancer gastrointestinal cancers
  • breast cancer breast cancer
  • prostate cancer and glioblastoma
  • GIST gastrointestinal stromal tumors
  • the tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, pazopanib and sunitinib.
  • the tyrosine kinase inhibitor is imatinib or dasatinib or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt of imatinib is imatinib mesylate.
  • the invention relates to combination/adjuvant therapy with a TKI and an isolated anti-WT-1 antibody, or antigen-binding portion thereof.
  • 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.
  • HC heavy chain
  • LC light chain
  • the WT-1 antibody or antigen-binding fragment thereof, comprises a V H and V L comprising first and second amino acid sequences, as shown in Tables 1 to 14 below and Figures 7-10.
  • the WT-1 antibody comprises the amino acid sequence of an scFv shown in Tables 1 to 14 below and Figures 7-10.
  • 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 D less than 1 X 10 "8 M; in one embodiment, the K D 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.
  • ADCC antibody dependent cellular cytotoxicity
  • Figure 1 shows that imatinib at 1 ⁇ , 5 ⁇ or 10 ⁇ does not affect antigen- dependent cellular cytotoxicity of human effector cells at various effector: target ratios (E:T) against fresh BV173 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.
  • target ratios E:T
  • HLA-A0201 + a leukemia cell line derived from CML in blastic crisis
  • 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.
  • Figure 3 shows luciferin imaging of BV173 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 ⁇ g anti-WT-1 /HLA-A antibody twice a week to mice with tumors. Control animals received neither imatinib nor antibody (upper left).
  • Figure 4 shows the effects of administration of ESKM and dasatinib at 1 ⁇ , 5 ⁇ or 10 ⁇ on ADCC of human effectors cells at various effector: target ratios (E:T) against BV173.
  • Figure 5 shows the effect of treatment with dasatinib alone or in
  • Figure 6 shows luciferin imaging of BV173 xenograft NSG mice after five weeks of therapy with a control treatment with dasatinib alone or in combination with an anti-WT-1 antibody.
  • Figures 7-10 show amino acid sequences, including consensus
  • Figure 11 shows the growth of BV173R following start of therapy on day 6 with tyrosine kinase inhibitors, Imatinib and Dasatinib, and anti-WT-1 /HLA0201 antibody (ESKM).
  • tyrosine kinase inhibitors Imatinib and Dasatinib
  • ESKM anti-WT-1 /HLA0201 antibody
  • Figure 12 shows the growth of BV173R in NSG mice treated with ESKM and Ponatinib: ⁇ control; ⁇ ESKM only; X Ponatinib only; >l ⁇ Ponatinib and ESKM.
  • Figure 13 shows luciferin imaging of BV173 xenograft NSG mice after five weeks of therapy.
  • ADCC Antibody-dependent cellular cytotoxicity
  • ALL Acute lymphocytic leukemia
  • AML Acute myeloid leukemia
  • CMC Complement mediated cytotoxicity
  • CDR Complementarity determining region (see also HVR below)
  • Ci_ Constant domain of the light chain
  • CH-i 1 st constant domain of the heavy chain
  • CML chronic myelogenous leukemia; also referred to as chronic myelocytic leukemia and chronic myeloid leukemia
  • FACS Fluorescence-activated cell sorting
  • FBS Fetal bovine serum
  • HLA Human leukocyte antigen
  • HVR-H Hypervariable region-heavy chain (see also CDR)
  • HVR-L Hypervariable region-light chain (see also CDR)
  • MHC Major histocompatibility complex
  • MM Multiple myeloma
  • TKI tyrosine kinase inhibitor
  • V H Variable heavy chain includes heavy chain hypervariable region and heavy chain variable framework region
  • V L Variable light chain includes light chain hypervariable region and light chain variable framework region
  • WT-1 Wilms tumor protein 1
  • administering and “administration” refer to the application of an active ingredient to the body of a subject.
  • 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.
  • VH heavy chain variable region
  • CH heavy chain constant
  • the heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant CL region.
  • the light chain constant region is comprised of one domain, CL.
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the
  • immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1 q) of the classical
  • 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 antigen) e.g., an antigen-binding protein
  • antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • antibody fragments encompassed within the term "antibody fragments" of an antibody include a Fab fragment, a monovalent fragment consisting of the V L , V H , C L 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 V L and V H domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a V H domain; and an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and V H regions pair to form monovalent molecules.
  • scFv single chain Fv
  • 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.
  • 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.
  • 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.
  • terapéuticaally 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
  • 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 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.
  • 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.
  • 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,
  • 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.
  • 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
  • Nilotinib (marketed as TASIGNA®) is approved to treat some patients with CML.
  • the drug is another small-molecule tyrosine kinase inhibitor.
  • TASIGNA® Prescribing Information [Novartis] (which is incorporated by reference in its entirety), lists recommendations for nilotinib administration and relevant data.
  • Bosutinib (marketed as BOSULIF®) is also approved to treat some patients with CML and is another example of a small-molecule tyrosine kinase inhibitor.
  • BOSULIF® Prescribing Information [Pfizer] (which is incorporated by reference in its entirety), lists recommendations for bosutinib administration and relevant data.
  • 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.
  • 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; Cabezon-Gutierrez 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
  • WT-1 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 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).
  • AML acute myeloid leukemia
  • WT- 1 For the most part, clinically approved therapeutic monoclonal antibodies (mAbs) (for example, trastuzumab) recognize structures of cell surface proteins.
  • WT- 1 is a nuclear protein and, therefore, is inaccessible to classical antibody therapy.
  • 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.
  • CTL cytotoxic CD8 T cell
  • 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 D of about 1 X10 "11 M to 1 X10 "8 M; (b) induces antibody dependent cellular cytotoxicity (ADCC) against WT-1 -expressing cells; or (c) inhibits growth of WT-1 positive cells in vivo.
  • 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.
  • RVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 54) WT-1 (Ext002 #13)
  • 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).
  • CDRs of other WT-1 antibodies suitable for use in practicing the disclosed method are shown in Figures 7-10. Table 1 3
  • 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).
  • 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).
  • the antibody is an anti -WT-1 /A2 antibody and comprises the human lgG1 constant region and Fc domain shown in Table 9.
  • 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.
  • Glycosylation (specifically fucosylation ) variants of IgG Fc can be produced using host expression cells and methods described in U.S. patents 8,025,879;
  • 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.
  • 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.
  • a method of treating cancer in a subject in need thereof is effected by administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor and a therapeutically effective amount of an anti-WT-1 antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • intramuscular subcutaneous and intramedullary injections
  • intrathecal direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • Methods for reducing the proliferation of leukemia cells comprising contacting leukemia cells with a WT-1 antibody of the invention.
  • the antibodies of the invention can be used for the prevention or treatment of leukemia. Administration of therapeutic antibodies is known in the art.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • Embodiments contemplated in view of the foregoing description include, but are not limited to, the following numbered embodiments:
  • 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
  • tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib.
  • a method for treating or inhibiting the proliferation of a WT-1 positive cancer 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.
  • 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.
  • CML chronic myelogenous leukemia
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • gastrointestinal stromal tumor ovarian cancer
  • prostate cancer soft tissue sarcoma
  • malignant glioma malignant glioma
  • tyrosine kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, erlotinib, gefitinib, lapatinib, sorafenib, and sunitinib.
  • 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 V H and V L comprising first and second amino acid sequences from Tables 1 -12; or
  • PBMC peripheral blood mononuclear cells
  • All cells were HLA typed by the Department of Cellular Immunology at Memorial Sloan-Kettering Cancer Center.
  • Leukemia cell line, BV173, (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 37° C/5% C0 2 .
  • BV173 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.
  • ADCC Antibody-dependent cellular cytotoxicity
  • 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 BV173 cell line, derived from CML in blastic crisis. Fresh BV173 cells were used for ADCC target cells. WT-1 antibody or its isotype control human lgG1 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 ⁇ . The supernatants were harvested and the cytotoxicity was measured by standard chromium 51 release assay.
  • WT-1 antibody mediated dose-dependent PBMC ADCC against naturally presented RMF epitope by HLA-A0201 molecule on tumor cells, the leukemia cell line BV173.
  • 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.
  • 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
  • BV173 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 BV173 with the resistant T315I Bcr-Abl plasmid.
  • ADCC Antibody-dependent cellular cytotoxicity
  • NSG NOD/SCID gamma
  • BLI bioluminescence imaging
  • 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 ⁇ g twice weekly IP.
  • ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro.
  • 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.
  • Miederer M et al. Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha particle therapy applications. Adv Drug Deliv f?ei 2008; 60 (12): 1371 -1382.
  • T-cell-receptor-like antibodies novel reagents for clinical cancer immunology and immunotherapy.
  • Cheever MA et al. The prioritization of cancer antigens: A national Cancer Institute pilot project for the acceleration of translational research. Clin Cancer Res 2009; 15 (17): 5323-5337.
  • Raphael A et al. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nature Medicine 2000; 6:443-446.

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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

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. §1 19(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 R01 CA 55349 and P01 CA 23766 awarded by the U.S. National Institutes of Health. The government has certain rights in the invention.
Sequence Listing
[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 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). [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 H LA- 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 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. [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 KD less than 1 X 10"8M; in one embodiment, the KD is in the range of about 1 X 10"11 M to about 1 X 10"8M. 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 μΜ, 5 μΜ or 10 μΜ does not affect antigen- dependent cellular cytotoxicity of human effector cells at various effector: target ratios (E:T) against fresh BV173 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. [0023] Figure 3 shows luciferin imaging of BV173 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 μg 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 μΜ, 5 μΜ or 10 μΜ on ADCC of human effectors cells at various effector: target ratios (E:T) against BV173.
[0025] Figure 5 shows the effect of treatment with dasatinib alone or in
combination with an anti-WT-1 antibody on BV173 tumor growth in NSG mice over four weeks of treatment.
[0026] Figure 6 shows luciferin imaging of BV173 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 BV173R following start of therapy on day 6 with tyrosine kinase inhibitors, Imatinib and Dasatinib, and anti-WT-1 /HLA0201 antibody (ESKM). 0 control;□ ESKM only; Δ Imatinib only; X Imatinib and ESKM; >l< Dasatinib only; O Dasatinib and ESKM.
[0029] Figure 12 shows the growth of BV173R in NSG mice treated with ESKM and Ponatinib:□ control; Δ ESKM only; X Ponatinib only; >l< 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 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] Ci_: Constant domain of the light chain
[0041 ] CH-i : 1 st constant domain of the heavy chain
[0042] CH!, 2, 3: 1 st, 2nd and 3rd constant domains of the heavy chain
[0043] CH2, 3: 2nd and 3rd constant domains of the heavy chain
[0044] CHO: Chinese hamster ovary [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] koft: 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 [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 system (e.g., effector cells) and the first component (C1 q) 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, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. These are known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
[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. [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
[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. [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 ICLUSIG™) 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; Cabezon-Gutierrez 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 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 KD of about 1 X10"11 M to 1 X10"8 M; (b) induces antibody dependent cellular cytotoxicity (ADCC) against WT-1 -expressing cells; or (c) inhibits 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 G 11 PI FGTANYAQKFQG RIPPYYGMDV
(SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4)
DNA ggaggcaccttcagcag gggatcatccctatctttggtac egg attcccccgtactacg gtat 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: 1 1 ) (SEQ ID NO: 13)
Full QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE VH WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY CARRIPPYYGMDVWGQGTTVTVSS (SEQ ID NO: 14)
DNA caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg cagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctgg ggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 15)
Full QTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL VL LIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD SLNGVVFGGGTKLTVLG (SEQ ID NO: 16)
DNA cagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgtt ctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcc cccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctcca agtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgt gcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg t (SEQ ID NO: 17) 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 )
DNA cgggcaagtcagagcatt gctgcatccagtttgcaaagt caacagagttacagtacccct agcagctatttaaat (SEQ ID NO: 30) ctcact (SEQ ID NO: 31 ) (SEQ ID NO: 29)
Full QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSN SAAWNWIRQSPSRGL VH EWLGRTYYGSKWYNDYAVSVKSRITINPDTSK NQFSLQLNSVTPEDTA VYYCARGRLGDAFDIWGQGTMVTVSS (SEQ I D NO: 32)
DNA caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgt gccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcg agaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctg tgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactct gtgactcccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctgg ggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 33)
Full DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY VL AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT FGGGTKVDIKR (SEQ ID NO: 34)
DNA gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgt (SEQ ID NO: 35)
scFv DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT
FGGGTKVDIKRSRGGGGSGGGGSGGGGSLEMAQVQLQQSGPGLVK
PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWY
NDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGRLGDAF
DIWGQGTMVTVSS
(SEQ ID NO: 36)
DNA gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgttctagaggtg gtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtac agctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatct ccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggc cttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctgtgaaaa gtcgaataaccatcaacccagacacatccaagaaccag ttctccctg cag ctg aactctg tg actc ccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctggggccaa gggacaatggtcaccgtctcttca (SEQ ID NO: 37) Table 3
WT-1 (Ext002 #13)
Antigen
Peptide F IMFPNAPYL (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) (SEQ ID NO: 49)
Full VH QMQLVQSGAEVKEPGESLRISCKGSGYSFTN FWISWVRQMPGKGLE
WMGRVDPGYSYSTYSPSFQGHVTISADKSTS )TA YLQW N S LKAS DTA MYYCARVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 50)
DNA cagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatctcct gtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaa ggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttccaag gccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaag gcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacc cctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 51 )
Full QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK VL LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGWVFGGGTKLTVLG (SEQ ID NO: 52)
DNA caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggt (SEQ ID NO: 53)
scFv QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK
LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD
DSLNGWVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQMQLV
QSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLEWMGR
VDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCA
RVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 54) WT-1 (Ext002 #13)
Antigen
Peptide RMFPNAPYL (SEQ ID NO: 1 )
DNA caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct aggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatgg cccagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatct cctgtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccggga aaggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttcca aggccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctga aggcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcga cccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 55)
Table 4
WT-1 (Ext002 #15)
Antigen
Peptide RMFPNAPYL (SEQ ID NO: 1 )
DNA gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO: 71 )
scFv DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI
YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT
FGQGTKVEIKRSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKK
PGESLKISCKGSGYNFSNKWIGWVRQLPGRGLEWIAIIYPGYSDITYSP
SFQGRVTISADTSINTAYLHWHSLKASDTAMYYCVRHTALAGFDYWGL
GTLVTVSS (SEQ ID NO: 72)
DNA gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgttctagaggtggt ggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtgcag ctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctgtaagggttct ggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagaggcctggagtg gatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggccgcgtcacc atctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggcctcggacac cgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgggcaccctggt caccgtctcctca (SEQ ID NO: 73)
Table 5
WT-1 (Ext002 #18)
Antigen
Peptide R FPNAPYL (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 gtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagctccagggaag gggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgagg ggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctg agagccgaggacacggccttgtattactgtgcgagagagcgtggctacgggtaccatgatccc catgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 87)
Full QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL VL VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW DSSSDHVVFGGGTKLTVLG (SEQ ID NO: 88)
DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct gaccgtcctaggt (SEQ ID NO: 89)
scFv QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL
VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW
DSSSDHVVFGGGTKLTVLGSRGGGGSGGGGSGGSLEMAEVQLVQ
SGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVS
GINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTALYY
CARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 90)
DNA cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct gaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggtggatccc tcgagatggccgaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtcc ctgagactctcctgtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaag ctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgca gactctgtgaggggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaa atgaacagtctgagagccgaggacacggccttgtattactgtgcgagagagcgtggctacggg taccatgatccccatgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID
NO: 91 ) Table 6
WT-1 (Ext002 #23)
Antigen
Peptide RMFPNAPYL (SEQ ID NO. 1 )
DNA cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta ggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag atggccgaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcaga ctctcctgtgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagg gaagggactggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgca gggccgattcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaa gccgaggacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctg gggccaaggcaccctggtgaccgtctcctca (SEQ ID NO. 109)
Table 7
Antigen WT-1 (Ext002B #17)
Peptide CMTWNQMNL (SEQ ID NO: 239)
CDRs: 1 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) Antigen WT-1 (Ext002B #17)
Peptide CMTWNQMNL (SEQ ID NO: 239)
DNA caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctg caaggtttccggatacaccctcactgaattatccatgcactgggtgcggcaggctcctggaaaagg gcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccaggg cagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgcgctctttctactcttactacggtatcgatacttggggtc aaggtactctggtgaccgtctcctca
(SEQ ID NO: 253)
Full SSELTQDPAVSVALGQTVRITCQGDSLRRYYASWYQQKPGQAPVLVI VL YANNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDISVN
GWMFGGGTKLTVLG
(SEQ ID NO: 254)
DNA tcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgcca aggagacagcctcagaaggtattatgcaagctggtaccagcagaagccaggacaggcccctgt acttgtcatctatgctaataacaatcggccctcagggatcccagaccgattctctggctccagctcag gaaacacagcttccttgaccatcactggggctcaggcggaggatgaggctgactattattgtaactc ccgggacatcagtgttaacggttggatgttcggcggagggaccaagctgaccgtcctaggt
(SEQ ID NO: 255)
scFv SSELTQDPAVSVALGQTVRITCQGDSLRRYYASWYQQKPGQAPVLVI
YANNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDISVN
GWMFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGA
EVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPED
GETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARSFYSY
YG I DTWGQGTLVTVSS
(SEQ ID NO: 256)
DNA tcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgcca aggagacagcctcagaaggtattatgcaagctggtaccagcagaagccaggacaggcccctgt acttgtcatctatgctaataacaatcggccctcagggatcccagaccgattctctggctccagctcag gaaacacagcttccttgaccatcactggggctcaggcggaggatgaggctgactattattgtaactc ccgggacatcagtgttaacggttggatgttcggcggagggaccaagctgaccgtcctaggttctag aggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggccc aggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgc aaggtttccggatacaccctcactgaattatccatgcactgggtgcggcaggctcctggaaaaggg cttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggc agagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagat ctgaggacacggccgtgtattactgtgcgcgctctttctactcttactacggtatcgatacttggggtca aggtactctggtgaccgtctcctca
(SEQ ID NO: 257) Table 8
Antigen WT-1 (Ext002B #28)
Peptide CMTWNQMNL (SEQ ID NO: 239)
scFv SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIY
YDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD
HSPYVFGTGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSG
SELKKPGASVKVSCKASGY I h I I YGMNWVRQAPGQGLEWMGWINTN
TGKPTYAQGFTGRFVFSLDASVSTAYLQISGLKADDTAVYYCARGYYG
WDYHD
(SEQ ID NO: 274)
DNA tcctatgtgctgactcagccaccctcagtgtcagtggccccaggaaagacggccaggattacctgt gggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccct gtgctggtcatctattatgatagcgaccggccctcagggatccctgagcgattctctggctccaactct gggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtc aggtgtgggatagtagtagtgatcattccccttatgtcttcggaactgggaccaagctgaccgtccta ggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag atggcccaggtgcagctggtgcagtctgggtctgagttgaagaagcctggggcctcagtgaaggtt tcctgcaaggcttctggatacaccttcactacctatggtatgaattgggtgcgacaggcccctggaca agggcttgagtggatgggatggatcaacaccaacactgggaagccaacgtatgcccagggcttc acaggacggtttgtcttctccttggacgcctctgtcagcacggcatatctgcagatcagcggcctaaa ggctgacgacactgccgtgtattactgtgcgcgcggttactacggttgggactaccatgattactggg gtcaaggtactctggtgaccgtctcctca
(SEQ ID NO: 275)
Table 9
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 cccaaactcatg atttatg atgtcagtgagcggccctcaggggtttctaatcgcttctccggctccaag tctggcaacacggccttcctgaccatctctgggctccaggctgaggacgaggctgattattactgca gctcatttgcagccagcagtccctggctgttcggcggagggaccaaggtcaccgtcctaggttcta gaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcc caggtgcagctggtggagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg cagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag atctgaggacacggccgtgtattactgtgcgcgctggttctacatgcaggctggtgatcattggggtc aaggtactctggtgaccgtctcctca
(SEQ ID NO: 293)
Table 10
Antigen WT-1 (Ext002B #40)
Peptide ( ITWNQMNL (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 g g attcaccttcag tag ct 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) Antigen WT-1 (Ext002B #40)
Peptide CMTWNQMNL (SEQ ID NO: 239)
DNA gaaattgtgctgactcagtctccactctccctgcccgtcacccttggacagccggcctccatctcctgc aggtctagtcaaagcctcgtatacagtgatggaaacacctatttgaattggtttcagcagaggccag gccaatctccaaggcgcctaatttatcaggtttctaagcgggactctggggtcccagacagattcag cggcagtgggtcaggcactgatttcacactgaaaatcagcagggtggaggctgaggatgttggga tgtattactgcatgcaaggttcacacttgcggacgttcggccaagggaccaaggtggaaatcaaac gttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagat ggcccaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgaggctct cctgtgcagcctctggattcaccttcagtagctatggcatgaactgggtccgccaggctccagggaa ggggctggagtgggtctcatccattagtagtagtagtagttacatatactacgcagactcagtgaag ggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgag agccgaggacacggctgtgtattactgtgcgcgcatccaggacgctactggtgaagaaatgatcct gtacgattactggggtcaaggtactctggtgaccgtctcctca
(SEQ ID NO: 31 1 )
Table 1 1
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 cag gtg tg g gttaatcctg at ggatttaatggtgttcat aatgaatatgtc
(SEQ ID NO: 321 ) (SEQ ID NO: 322) (SEQ ID NO: 323)
Full QVQLVQSGAEVK <PGASVKVSCKASGYTFTDYYI HWVRQAPGQGLEWM VH GWMNPNSGNSVS SAQKFQGRVTMTRDTSINTAYIV IELSSLTSDDTAVYYC
ARYQGSTWKYDSYGDLWGQGTLVTVTS
(SEQ ID NO: 324) 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 caggctgtgctgactcagccaccctcggtgtcagtggccccaggagagacggccactgttacctgtgg gggaaacgagattggatttaatggtgttcattggtataagcagaaggcaggccaggcccctctgttggtc atctataacaatagggtccggccctcagggatctctgagcgactctctggctccaactctggtaacacgg ccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggttaat cctgataatgaatatgtcttcggatcggggaccaaggtcaccgtcctaggt
(SEQ ID NO: 327)
scF QAVLTQPPSVSVAPGETATVTCGGNEIGFNGVHWYKQKAGQAPLLVIYN
V NRVRPSGISERLSGSNSGNTATLTISRVEAGDEADYYCQVWVNPDNEYV
FGSGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKP
GASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWMNPNSGNSVSA
QKFQGRVTMTRDTSINTAYMELSSLTSDDTAVYYCARYQGSTWKYDSY
GDLWGQGTLVTVTS
(SEQ ID NO: 328)
DNA caggctgtgctgactcagccaccctcggtgtcagtggccccaggagagacggccactgttacctgtgg gggaaacgagattggatttaatggtgttcattggtataagcagaaggcaggccaggcccctctgttggtc atctataacaatagggtccggccctcagggatctctgagcgactctctggctccaactctggtaacacgg ccaccctgaccatcagcagggtcgaagccggggatgaggccgactattactgtcaggtgtgggttaat cctgataatgaatatgtcttcggatcggggaccaaggtcaccgtcctaggttctagaggtggtggtggt agcggcggcggcggctctggtggtggtggatccctcgagatggcccaggtccagctggtgcag tctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttc accgactactatatacactgggtgcggcaggcccctggacaagggctggagtggatgggatggatga accctaacagtgggaactcagtctctgcacagaagttccagggcagagtcaccatgaccagggatac ctccataaacacagcctacatggagctgagcagcctgacatctgacgacacggccgtatattactgtgc gcgctaccagggttctacttggaaatacgactcttacggtgatctgtggggtcaaggtactctggtgacc gtcacctca
(SEQ ID NO: 329) Table 12
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. Table 1 3
CDR-H1 CDR-H2 CDR-H3 SEQ ID
NO.
Group I
EXT002-12(166) SNAVAWN RTYRGSTYY ALSV G-SNSAFDF 119-121
EXT002-5(184) SNSAAWN RTYYGSKWYNDYAVSV GRLGDAFDI 122-124
EXT002-8(184) SDGAAWN RTYYRSKWYNDYAVSV GDYYYGMDV 125-127
Consensus(191) SNAAAWN RTYYGSKWYNDYAVSV G AFDI 128-130
Group II
EXT002-14(163) SYWIS RIDPSDSYTNYSPSFQG GD YDFYLDP— 131-133
EXT002-25(163) SYGIS WI SAYNGNTNYAQKLQG DLYSSGWYESYYYGMDV 134-136
EXT002-3(186) SYAIS GIIPIFGTANYAQKFQG RIP-P YYGMDV 137-139
EXT002-30(163) SYGIS WI SAHNGNTNYAQKLQG DR VWFGDLSD 134, 140
141
EXT002-33(163) SYAIS GIIPIFGTANYAQKEQG NYDFWSG DAFDI 137, 142
143
Consensus(188) SYAIS I P G TNYAQKFQG FY GMDV 137, 144
145
Group III
EXT002-34(161) DYGMS GINWNGGSTGYADSV ERGY-GYHDPHDY 146-148
EXT002-40(157) NYTMN SISLSGAYIYYADSL EGYSSSVYDAFDL 149-151
EXT002-45(165) SYGMH GILSDGGKDYYVDSV CSSN-YGNDAFDI 152-154
EXT002-48(165) TYSMN SISSGAYSIFYADSV DQYYGDKWDAFDI 155-157
Consensus (170) SYGMN SISS GGS IYYADSV E YY WDAFDI 158-160 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, 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) RASQGI SNYLA AASTLQS QKYNSAPGVT 201-203
EXT002-35 (24) RASQS INGWLA RASTLQS QQSSSLP-FT 204-206
EXT002-5 (48) RASQSISSYLN AASSLQS QQSYSTP-LT 198-200
EXT002-7 (48) RASQGI SYYLA 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 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
Table 15
Constant Regions
Human heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL constant region and TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT IgGl Fc domain KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR sequence TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK (SEQ ID NO. 236)
Human light chain TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL (kappa) QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC (SEQ ID NO. 237)
Human light chain QPKANPTVTLFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADGS (lambda)
PVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG STVEKTVAPTECS (SEQ ID NO. 238)
[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 lgG1 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 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 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 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 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. [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] 1 1 . 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 (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 KD of 1 X10"8 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 KD of about 1 X10"11 M to about 1 X10"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 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, BV173, (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 37° C/5% C02.
[00131] Animals. Six to eight week-old male NOD.Cg-Prkdc scid IL2rgtm1 Wjl/SzJ mice, known as NOD/SCI D 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. BV173 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 BV173 cell line, derived from CML in blastic crisis. Fresh BV173 cells were used for ADCC target cells. WT-1 antibody or its isotype control human lgG1 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 μΜ. The supernatants were harvested and the cytotoxicity was measured by standard chromium 51 release assay. [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 BV173. 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 BV173 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 μg 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. [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 μg 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 VHLA antibody (ESKM) in a human leukemia xenograft NSG model. Three million BV173 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 μg 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 μί. 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 1 1 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 received only dasatinib initially had clearance of tumor, but relapsed by week 4 of therapy. Animals that received 100 μ9 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] BV173 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 BV173 with the resistant T315I 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 μg twice weekly IP.
[00141] ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro. In a BV173 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 T315I BV173- R leukemia in vivo. Cures were not achieved with combination therapy of ESKM and either first or second generation TKIs against resistant T315I 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 reduction (Figure 13). The conclusion is that ESKM is an effective therapeutic antibody for sensitive and T315I Ph+ ALL. Resistant T31 5I 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

CLAIMS What is claimed is:
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, eriotinib, 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 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.
1 1 . 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.
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 KD of 1 X10"8 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 KD of about 1 X10"11 M to about 1 X10"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).
EP14722878.7A 2013-03-15 2014-03-14 Combination/adjuvant therapy for wt-1-positive disease Withdrawn EP2968539A1 (en)

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