CN106170297A - Combination/complementary therapy for WT 1 positive diseases - Google Patents

Abstract

In order to improve primary disease response and/or overcome resistance disease, the disclosure provides a kind of method of propagation for treating or suppress WT 1 dependence cancer, described method includes providing the tyrosine kinase inhibitor of therapeutically effective amount and anti-WT 1/HLA antibody to experimenter in need, i.e. the antibody of the peptide of Wei Ermusishi oncoprotein (WT 1) to present on the specific binding cancer cell surfaces of HLA restrictive one.

Description

Combination/complementary therapy for WT-1-positive diseases

Cross-Reference to Related Applications

The application is containing the theme relevant to the theme of documents below: in the entitled " T-Cell that on April 2nd, 2012 submits to Receptor Like Antibodies Specific for WT1Peptides " commonly assigned PCT international application sequence Number PCT/US2012/031892 and entitled " the Antibodies to Cytosolic submitted on March 15th, 2013 Peptides " the commonly assigned U.S. Provisional Application No. 61/798,563 of (attorney 3314.030P)；In each document Hold and be hereby incorporated herein in its entirety by reference.

Hereby give notice that in the U.S. Provisional Patent Application Serial No. 61/794,168 of submission on March 15th, 2013 with in 2013 The U.S. Provisional Patent Application Serial No. 61/880,585 of on JIUYUE submission in 20, rights and interests under 35U.S.C. § 119 (e), and The disclosure of two priority documents is all incorporated herein in its entirety by reference.

Federal government subsidizes the rights statement of research

The present invention completes under governmental support, NIH provide NIH R01CA 55349 and P01CA No. 23766 funds are subsidized.Government has certain rights in the invention.

Sequence table

The application is contained in the sequence table set up on March 14th, 2014；File is ASCII fromat, is named as 48319_ SeqListing.txt and be 182,226 bytes.This document is incorporated hereby the application hereby.

Technical field

The present invention relates generally to the treatment of WT-1-positive diseases such as chronic lymphocytic leukemia (CML).More specifically, this Invention relates to the suppression of tumor growth and utilizes treatment with tyrosine kinase inhibitors agent and for Wei Ermusishi tumor cancer base Combined therapy because of the antibody of albumen (WT-1).

Background of invention

Up to now, the treatment of cancer (such as CML) relies on the therapeutic agent with protein tyrosine kinase as target.Tyrosine-kinase Enzyme inhibitor (TKI) comprises imatinibDasatinibSutent, Suo Lafei Buddhist nun, pazopanib, name just a few.Tyrosine kinase inhibitor is currently the first-line treatment agent in following disease treatment: chronic bone Marrow (also referred to as marrow or myeloid) leukemia (CML), acute myeloid leukaemia (AML), Acute Lymphoblastic Leukemia (ALL) and myelodysplastic syndrome (MDS), ovarian cancer, carcinoma of prostate, soft tissue sarcoma, glioblastoma, Renal cell carcinoma, hepatocarcinoma, gastrointestinal stromal tumor (GIST), breast carcinoma, pulmonary carcinoma etc..But, some TKI (such as, imatinib And Sutent) clinical efficacy, be limited to rare patient-specific medication's Intolerance or treatment refractory disease send out Exhibition.

Except the little molecule therapy of targeting protein tyrosine kinase, developing based on using vaccine and tumour-specific to resist The leukemia treating of the immunization method of body.Such as, Wei Ermusishi tumor oncogene protein (WT-1) has become as mostly The attractive target of the immunotherapy of number leukemia (including CML and various cancer).WT-1 is during embry ogenesis The zinc finger transcription factor of normal expression in mesoderm tissues.In normal adult tissue, WT-1 expresses at CD34⁺Hematopoietic Stem is thin Born of the same parents are limited in low-level, but process LAN (1-2) in the leukemia and various entity tumor of multiple pedigree.Recently, according to report Road WT-1 is expressed as the mark of minimum residual disease.Increase in acute myeloid leukaemia (AML) patient that morphology is alleviated turns The most predicted obvious clinical recurrence of record level (3,4).Additionally, detect in Hematopoietic Malignancies and solid tumor patient The antibody for WT-1 arrived, shows that WT-1 is high immunogenicity antigen (7).

In most of the cases, the therapeutic monoclonal antibodies (mAb) of clinical approval identifies the structure of cell surface protein. But, WT-1 is nucleoprotein, and is therefore that classical antibody therapy institute is unapproachable.Up to date, the immunity of targeting WT-1 Therapy has been limited to cellular processes, and cellular processes purpose is only that generation WT-1-specific cytotoxicity cd8 t cell (CTL) Response, the peptide that described response identification is presented on cell surface by MHC I quasi-molecule.

In order to induce CTL response, generally decomposed intracellular protein by proteasome or inscribe/lysosome, and obtained Fragments of peptides combine MHC I class or II quasi-molecule.These peptides-MHC complex cell surface show, wherein they by peptide- MHC (pMHC)-φt cell receptor (TCR) interacts provides the target (8,9) of T cell identification.Utilization derives from WT-1 albumen Peptide vaccination induction HLA restrictive cell toxicity cd8 t cell, it can kill tumor cell.

Other cancer treatment method utilizes monoclonal antibody therapy target on cancer antigen.Show monoclonal antibody (mAb) Therapy plays the strongest Graft Versus Tumor by number of mechanisms, and described mechanism includes CDC (CDC), resists Body dependent cellular cytotoxicity (ADCC) and act on the direct Carbazole alkaloid of tumor cell or the apoptosis of process LAN target molecule Inductive effect.

If there is improving primary disease response, overcome resistance disease and/or reduce the effective of monotherapy agent , will there is great benefit, such as in the auxiliary treating method of dosage, it is to avoid the toxicity of TKI and other adverse side effect.

Summary of the invention

The disclosure provides a kind of method for treating WT-1 positive diseases, and described method is based on orientation different molecular target The combination of therapeutic agent.Described method is incorporated to use tyrosine kinase inhibitor (TKI) (as orientation Bcr-Abl (her horse For Buddhist nun and Dasatinib), and orient the TKI (such as erlotinib and gefitinib) of other molecular target (such as EGFR)) normal Rule treatment, and based on using with HLA restrictive one identification the immunization therapy side of the antibody of the peptide combining WT-1 cancer protein Method.

The present invention is based on beyond thought observation, i.e. when compared with being administered alone, by TKI and anti-WT-1 antibody group The therapeutic scheme closed causes early stage suppression and the Anti-tumor response of enhancing of tumor growth.In some embodiments, TKI The usage amount of TKI is allowed to be less than the amount utilized in treating above-mentioned condition of illness at present, simultaneously with jointly using of anti-WT-1 antibody Maintain the curative effect of TKI, and again while improve tumour progression time, total survival period and reduce the side effect relevant to TKI.

Therefore, in one aspect, the present invention relates to a kind of life of WT-1 positive cancer for treating or suppress experimenter Long method, described method is anti-by the separation of the tyrosine kinase inhibitor of administering therapeutic effective dose and therapeutically effective amount WT-1 antibody or its antigen-binding portion thereof, i.e. the antibody of the specific binding WT-1 peptide being combined with MHC antigen.Tyrosine kinase Inhibitor can be with oriented molecule target, such as Bcr-Abl (imatinib, Dasatinib and AMN107), EGFR (erlotinib And gefitinib), VEGFR-1 (pazopanib and Sorafenib) etc..

In one aspect, WT-1 positive cancer is selected from the group that consists of: chronic lymphocytic leukemia (CML), multiple Property myeloma (MM), acute lymphoblastic leukemia (ALL), acute myelogenous/myelomatosis (AML), myelosis Abnormal syndrome (MDS), mesothelioma, ovarian cancer, human primary gastrointestinal cancers, breast carcinoma, carcinoma of prostate and glioblastoma, gastrointestinal stromal tumor And include other tumor of solid tumor (GIST).

In one aspect, tyrosine kinase inhibitor is selected from the group consisted of: imatinib, Dasatinib, Ni Luo For Buddhist nun, SKI-606, Ponatinib, Ba Fei for Buddhist nun, erlotinib, gefitinib, Lapatinib, Sorafenib, pazopanib And Sutent.In one embodiment, tyrosine kinase inhibitor be imatinib or Dasatinib or its pharmaceutically may be used The salt accepted.In one embodiment, the pharmaceutically acceptable salt of imatinib is imatinib mesylate.

In yet another aspect, the present invention relates to use TKI and the anti-WT-1 antibody of separation or the group of its antigen-binding portion thereof Conjunction/complementary therapy.Example for the anti-WT-1 antibody with the combination treatment of TKI includes but not limited to:

Comprising the anti-WT-1 antibody of heavy chain (HC) variable region and light chain (LC) variable region, described heavy chain (HC) variable region is wrapped Containing HC-CDR1, HC-CDR2 and HC-CDR3, and light chain (LC) variable region comprises LC-CDR1, LC-CDR2 and LC-CDR3, institute State heavy chain (HC) variable region and light chain (LC) variable region and comprise the aminoacid sequence as shown in table 1 below to 14 and Fig. 7-10.

In yet another aspect, WT-1 antibody or its Fab comprise V_HAnd V_L, described V_HAnd V_LComprise such as hereafter table The first aminoacid sequence shown in 1 to 14 and Fig. 7-10 and the second aminoacid sequence.In yet another aspect, WT-1 antibody comprises Table 1 below is to the scFv aminoacid sequence shown in 14 and Fig. 7-10.

Disclosed method is adopted as the WT-1 antibody of fully human antibodies；Described antibody comprises people's variable domain framework region and people Constant region.WT-1 antibody with HLA restrictive one specificity with less than 1 × 10^-8The K of M_DIn conjunction with WT-1 peptide；An embodiment party In case, K_DAbout 1 × 10^-11M to about 1 × 10^-8In the range of M.WT-1 antibody induction is for the antibody-dependant of WT-1 positive cell Sexual cell toxicity (ADCC).

Accompanying drawing is sketched

Fig. 1 illustrates that the imatinib of 1 μM, 5 μMs or 10 μMs does not affect people effector lymphocyte at different effect: target compares ratio (E:T) for the antigen-dependent cytotoxity of fresh BV173 cell under, described fresh BV173 cell derived is in initiation potential CML Leukemia Cell Lines (the HLA-A0201 of phase⁺, Philadelphia Chromosome Positive).Change effector with target ratio to prove ESKM ADCC is for the dependency of high E:T ratio.

Fig. 2 illustrates the effect of anti-WT-1/HLA-A antibody, and named ESKM, for tumor growth every 13,20,27,34 Used with 40 days and do not use imatinib.

Fig. 3 is shown in and uses 50mg/kg imatinib five weeks to the mice of band tumor is daily, uses and (bottom-right graph) and not Use (lower-left figure) 100 μ g anti-WT-1/HLA-A antibody twice a week after, the fluorescein of BV173 xenotransplantation NSG mice becomes Picture.Control animal accepts imatinib or antibody (the picture left above).

Fig. 4 illustrates that the Dasatinib of ESKM and 1 μM, 5 μMs or 10 μMs is sub in different effect to people effector lymphocyte: target is frequently For the application effect of ADCC of BV173 under rate (E:T).

Fig. 5 illustrate Dasatinib individually or with the BV173 of the anti-WT-1 Antibody Combination NSG mice to exceeding surrounding treatment The therapeutic effect of tumor growth.

Fig. 6 illustrate Dasatinib individually or with the anti-WT-1 Antibody Combination control treatment treatment of 5 weeks after BV173 xenogenesis Transplant the fluorescein imaging of NSG mice.

Fig. 7-10 illustrates aminoacid sequence, comprises consensus sequence, for the CDR of some embodiment of anti-WT-1 antibody, Described anti-WT-1 antibody can be used for the combination treatment of the disclosure.

Figure 11 illustrates the growth of BV173R after starting to treat 6 days, and described treatment uses tyrosine kinase inhibitor, and she replaces by horse Buddhist nun and Dasatinib, and anti-WT-1/HLA0201 antibody (ESKM) ◇ comparison；Only ESKM；Δ only imatinib；X she replace by horse Buddhist nun and ESKM；>l<only Dasatinib；O Dasatinib and ESKM.

Figure 12 illustrates with ESKM and the growth of the BV173R of the NSG mice of Ponatinib treatment.Compares；Δ only ESKM；X Only Ponatinib；>l<Ponatinib and ESKM.

Figure 13 illustrates the fluorescein imaging of the BV173 xenotransplantation NSG mice after treating five weeks.

Detailed Description Of The Invention

Herein cited all publications, patent and other list of references are incorporated hereby the disclosure. The theme being incorporated by reference is not qualified as the alternative that any claim limits, unless otherwise expressly stated.

When putting into practice the present invention, employing the routine techniques in many immunologys, described technology is at those of ordinary skill In technical scope.These technology are described in greater detail in, such as, and " Curr ent Protocols in Immunology " (John E.Coligan etc. compile, John Wiley&Sons, Inc.1991 and regular update)；Recombinant Antibodies for Immunot herapy, Melvyn Little edits, Cambridge University Press 2009.Containing standard scheme, widely known for those skilled in the art and rely on, include description and the dosage information of manufacturer These lists of references and the content of other list of references be hereby herein incorporated by reference as a part of this disclosure.This Shen Please full a piece use following abbreviations:

Ab: antibody

ADCC: antibody dependent cellular cytotoxicity

ALL: acute lymphoblastic leukemia

AML: acute myeloid leukaemia

CDC: CDC

The cytotoxicity of CMC: complement-mediated

CDR: complementary determining region (referring also to hereafter HVR)

C_L: the constant region of light chain

CH₁: the first constant region of heavy chain

CH_1,2,3: first, second, and third constant region of heavy chain

CH_2,3: second and the 3rd constant region of heavy chain

CHO: Chinese hamster ovary

CML: chronic lymphocytic leukemia；Also chronic myelocytic leukemia and chronic myelogenous leukemia are referred to

CTL: cytotoxic T cell

E:T ratio: effector: target compares ratio

Fab: antibody binding fragment

FACS: Fluorescence-activated cell sorting

FBS: hyclone

FR: framework region

HC: heavy chain

HLA: human leucocyte antigen (HLA)

HVR-H: heavy chain hypervariable region (referring also to CDR)

HVR-L: light chain hypervariable region (referring also to CDR)

Ig: immunoglobulin

K_D: dissociation constant

k_off: dissociation rate

k_on: association rate

MHC: MHC

MM: multiple myeloma

ScFv: single chain variable fragment

TKI: tyrosine kinase inhibitor

V_H: comprise heavy chain hypervariable region and the variable heavy chain of weight chain variable framework region

V_L: comprise light chain hypervariable region and the variable light of light chain variable framework region

WT-1: Wei Ermusishi oncoprotein 1

In the following description, term used herein is intended to those terms as they are by those skilled in the art institute Known implication is as one man explained.Definition presented below is to illustrate rather than limit defined term.

As used herein, " using (administering/administration) " refers to that active component is to experimenter's body The application of body.

As those terms known in the art, " antibody " refers to immune antigen-binding proteins.As mentioned above Term " antibody " include complete, the full length antibody with antigen binding domain, and its retain described " antigen-binding portion thereof " or Any fragment of " antigen binding domain ", or its strand, such as single chain variable fragment (scFv).Naturally occurring " antibody " is to comprise At least two weight (H) chain interconnected by disulfide bond and the glycoprotein of two light (L) chains.Each heavy chain is by variable region of heavy chain (herein It is abbreviated as V_H) and light chain constant (CH) district composition.CH comprises three domains: CH1, CH2 and CH3.Each light chain by V (is abbreviated as herein in variable region of light chain_L) and chain constant C_LDistrict forms.Constant region of light chain comprises a domain, C_L。V_HAnd V_L District can be further subdivided into has denatured district, referred to as complementary determining region (CDR), is interleaved with more conservative district, referred to as frame Frame district (FR).Each V_HAnd V_LIt is made up of three CDR and four FR, is arranged to c-terminus by aminoterminal in the following order: FR1, CDR1、FR2、CDR2、FR3、CDR3、FR4.The binding structural domain with AI is contained in the variable region of heavy chain and light chain. The constant region of antibody can combine host tissue or the factor with mediated immunity globulin, including immune various cells (such as Effector lymphocyte) and first composition (C1q) of classical complement system.

As used herein, " antigen-binding portion thereof " or " antigen binding domain " of term antibody refers to the conjugated antigen of antibody also And give the antibody district with antigenic specificity or part；The fragment of antigen-binding proteins, such as, the reservation that antibody comprises antibody is special One or more fragments of the ability of anisogamy antigen (such as, peptide/HLA complex).Having shown that can be by the sheet of full length antibody The antigen combined function of Duan Zhihang antibody.The example of the Fab being covered by " antibody fragment " of term antibody includes: Fab fragment, it is by V_L、V_H、C_LMonovalent fragment with CH1 domain composition；F(ab)₂Fragment, it is for comprising by hinge region The bivalent fragment of two Fab fragments that connects of disulfide bond；By V_HFd fragment with CH1 domain composition；By the single armed of antibody V_LAnd V_HThe Fv fragment of domain composition；By V_HDAb fragment (Ward etc. (1989) the Nature 341:544-of domain composition 546)；And the complementary determining region (CDR) separated.

Although additionally, the two of Fv fragment domains, V_LAnd V_H, by single gene code, but recombination method can be used By allowing them to the synthetic linker becoming single protein chain, they are connected, in described single protein chain, V_LAnd V_HDistrict matches To form monovalent molecule.These are referred to as scFv (scFv)；See for example, Bird etc., 1988Science242:423-426； With Huston etc., 1988Proc.Natl.Acad.Sci.85:5879-5883.These antibody fragments are to use people in the art The known routine techniques of member obtains, and screens described fragment for effectiveness in the way of identical with complete antibody.

" antibody of separation " is intended to from its natural environment Components identification and separation and/or the antibody of recovery, with And " synthetic antibody " or " recombinant antibodies ", i.e. generally use recombinant technique well known by persons skilled in the art or use peptide symthesis skill Antibody produced by art.

As used herein, term " effective dose " means the group sought by initiation (such as, research worker or clinicist) Knit, system, animal or the biology of people or the compound of medical science response or the amount of therapeutic agent.

Term " therapeutically effective amount " means arbitrary such amount: compared to not receiving the corresponding experimenter of this dosage, Described amount makes the improved treatment of disease, disease or side effect, cures, prevents or improve, or disease or the evil of disease Rate reduces.Described term also includes the amount effectively strengthening normal physiological function in the range of it.

The present invention provides a kind of by jointly using tyrosine kinase inhibitor and anti-WT-1 Antybody therapy WT-1 positive disease Sick improved method.

Tyrosine kinase inhibitor

Tyrosine kinase inhibitor, and the treatment for some cancer that considers of route of administration and suitable dosage is ability Known to territory.These small-molecule drug targeting are referred to as protein-based some members of tyrosine kinase, described tyrosine kinase Participate in signal transduction.These enzymes overactivity in certain cancers, causes uncontrolled growth.

The tyrosine kinase inhibitor being applicable to method of disclosure includes: imatinib, Dasatinib, AMN107, ripple Relax for Buddhist nun, Ponatinib；With Ba Fei for Buddhist nun's imatinib, Dasatinib, AMN107, SKI-606, Ponatinib；And Ba Fei For Buddhist nun, erlotinib, gefitinib, Lapatinib, Sorafenib and Sutent.Following table provides some TKI, their molecule The list of the indication of target and FDA-approval.

Table

Imatinib mesylate (is marketed as) be approved for treating gastrointestinal stromal tumor (GIST, a kind of rare gastrointestinal cancer) and other mesenchymal neoplasm, Ph⁺CML, some other type of leukemia, protuberantia Dermatofibrosarcoma, myeloproliferative disorder/bone marrow proliferative condition of illness and systemic mastocytosis.Imatinib generally quilt It is considered as the first generation Bcr-Abl tyrosine kinase inhibitor for treating such as CML.Prescription information [2013:Novartis] (being incorporated hereby) lists the suggestion and related data used for imatinib.

Dasatinib (trade name) being approved for treating some, to suffer from CML or acute one-tenth lymph thin The leukemic patient of born of the same parents' property.Described medicine is the micromolecular inhibitor of some tyrosine kinase.Prescription information [Bristol-Myers Squibb] (being incorporated hereby) lists the suggestion and phase used for Dasatinib Close data.

AMN107 (trade name) be approved for treating some patients suffering from CML.Described medicine Thing is another kind of small molecule tyrosine kinase inhibitors.Prescription information [Novartis] is (by reference It is integrally incorporated) list the suggestion and related data that AMN107 is used.

SKI-606 (trade name) be also approved for treating some patients suffering from CML and being Another example of small molecule tyrosine kinase inhibitors.Prescription information [Pfizer] is (by reference It is integrally incorporated) list the suggestion and related data that Bosutinib is used.

Ponatinib (trade name ICLUSIG^TM) it is FDA approval, candidate is used for treating CML and Philadelphia Chromosome Positive Acute lymphoblastic leukemia (Ph⁺ALL) oral drugs.Ponatinib is many targeting TKI, but Ponatinib is main Target is BCR-ABL, and this abnormal tyrosine kinase is CML and Ph⁺The mark of ALL.

The prescription information of each therapeutic agent listed herein is incorporated herein in its entirety by reference.Press down about tyrosine kinase The dosage of preparation and/or the other information of side effect can find in the following documents: G.D.Demetri, Differential properties of current tyrosine kinase inhibitors in gastrointestinal stromal tumors；Warnault P etc., Recent Advances in Drug Design of Epidermal Growth Factor Receptor Inhibitors；Sivendran S etc., Treatment-related mortality with vascular endothelial growth factor receptor tyrosine kinase inhibitor therapy in patients with advanced solid tumors:a meta-analysis；Cabezón-Gutiérrez 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 below list of references 69-74)

Anti-WT-1 antibody

Wei Ermusishi tumor oncogene protein (WT-1) has become as most of leukemia and various cancer The attractive target of immunotherapy.WT-1 be during embry ogenesis in mesoderm tissues the zinc finger transcription of normal expression The factor.In normal adult tissue, WT-1 expresses at CD34⁺Hematopoietic stem cell is limited in low-level, but in various kinds of cell Process LAN (1-2) in the leukemia of pedigree and various entity tumor.Recently, it was reported that WT-1 is expressed as the mark of minimum residual disease Will.The most predicted obvious clinical recurrence of transcriptional level increased in acute myeloid leukaemia (AML) patient that morphology is alleviated (3,4).Additionally, the antibody for WT-1 detected in Hematopoietic Malignancies and solid tumor patient, show WT-1 It is high immunogenicity antigen (7).

In most of the cases, the therapeutic monoclonal antibodies (mAb) (such as, trastuzumab) ratified clinically identifies thin The structure of cellular surface albumen.But, WT-1 is nucleoprotein, and is therefore that classical antibody therapy institute is unapproachable.Until Closely, the immunotherapy of targeting WT-1 has been limited to cellular processes, and cellular processes purpose is only that generation WT-1-specific cell Toxicity cd8 t cell (CTL) response, the peptide that described response identification is presented on cell surface by MHC I quasi-molecule.

In order to induce CTL response, generally decomposed intracellular protein by proteasome or inscribe/lysosome, and obtained Peptide fragment combine MHC I class or II quasi-molecule.These peptides-MHC complex cell surface show, wherein they by peptide- MHC (pMHC)-φt cell receptor (TCR) interacts provides the target (8,9) of T cell identification.Utilization derives from WT-1 albumen Peptide vaccination induction HLA restrictive cell toxicity cd8 t cell, it can kill tumor cell.

It has been established that anti-WT-1 antibody can carry with jointly using of small molecule tyrosine kinase inhibitors The curative effect of high small molecule therapy agent.

The anti-WT-1 antibody bag that may be used for cancer combination treatment in the range of method and composition required for protection Include but be not limited to HLA restrictive one specific binding WT-1 peptide and show further in following characteristic at least one Those anti-WT-1 antibody: (a) is with about 1 × 10^-11M to 1 × 10^-8The K of M_DIn conjunction with WT-1/HLA；B () induction is expressed for WT-1 The antibody dependent cellular cytotoxicity (ADCC) of cell；Or (c) suppresses the growth of internal WT-1 positive cell.In some embodiments In, treat that using the anti-WT-1 antibody of pairing with TKI is to comprise to list in table 1-14 and one or more shown in Fig. 7-10 Those antibody of aminoacid sequence (scFv, VH and VL district or CDR).

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

In sequence in table 1-14, boldface type instruction height becomes sequence of heavy chain and height and lightens the connection sequence between chain-ordering Row.

In some embodiments, the anti-WT-1 antibody used in the methods of the invention also can be contained and comprise light chain and weight Shown in those of chain hypervariable region and constant region, such as table 13 (heavy chain), 14 (light chains) and 15 (constant regions).Similarly, The CDR being applicable to put into practice other WT-1 antibody of disclosed method illustrates at Fig. 7-10.

Table 13

Table 14

Table 15

In some embodiments, anti-WT-1 antibody is that wherein constant region/framework region is such as changed by aminoacid replacement Those become, with the characteristic of modified antibodies (such as, be increased or decreased following in one or more: antigen-binding affinity, Fc receptor combines, antibody carbohydrate, and such as, glycosylation, fucosylation etc., the number of cysteine residues, effect are thin Born of the same parents' function, effector cell function, the supplementary functions of binding site or introducing).

In one embodiment, antibody be anti-WT-1/A2 antibody and comprise the human IgG1 constant region shown in table 9 and Fc domain.In one embodiment, anti-WT-1/A2 antibody comprises people's κ sequence with sequence described in table 9, or people's λ sequence Row.The aminoacid sequence of some complementary determining regions (CDR) of antibody of the present invention is shown in table 1-14 and Fig. 7-10.

Can use host expression cell and method to produce glycosylation (particularly fucosylation) variant of IgG Fc, Described method is described in United States Patent (USP) 8,025,879；8,080,415；With 8,084,022, its content is herein incorporated by reference. In short, it is disclosed herein to obtain coding by the separation using the isolated and purified standard technique of RNA and be optionally based on size The heavy chain of antibody or the messenger RNA (mRNA) of light chain.Then techniques known in the art is used to produce and separate corresponding to coding The cDNA of the mRNA of heavy chain or light chain, described technology such as cDNA library builds, phage library builds and uses specific phase Close screening or the RT-PCR of primer.In some embodiments, cDNA sequence can be to use known external DNA operating technology The sequence of all or part of cDNA sequence manufactured to produce specific needs.Then cDNA sequence can be positioned in carrier, Promoter that described carrier contains together with gene in reading frame and compatible with the host cell that low fucose is modified.

One aspect of some embodiments according to the disclosure, it is provided that treat cancer in experimenter in need Method.According to these embodiments, described method by experimenter's administering therapeutic effective dose tyrosine kinase inhibitor and The anti-WT-1 antibody of therapeutically effective amount realizes.

As used herein, the development, substantially including eliminating, substantially suppress, slow down or reverse condition of illness " treated " in term Improve clinic or the aesthetical symptoms of condition of illness or be essentially prevented from the clinic of condition of illness or the appearance of aesthetical symptoms, and including, such as, Reduce the size of experimenter's tumor, affect the relieved state of experimenter, improve expection survival probability, increase life expectancy, with And increase the expeced time of progression of disease.

As described in subsequent embodiment part, surprisingly, when using together, it was observed that tyrosine kinase Inhibitor (such as imatinib and Dasatinib) and anti-WT-1 antibody have useful additive effect.Sand is reached it is essential that use Show the disease having cured them for some animals of Buddhist nun and anti-WT-Antibody Combination, but use the animal of arbitrary drug alone No.

The applicable route of administration of TKI can (such as) include being administered orally, rectum, through mucous membrane, especially per nasal, intestinal or parenteral Deliver, including in intramuscular, subcutaneous and intramedullary injection and sheath, in direct ventricle, intravenous, intraperitoneal, intranasal or ophthalmic Injection.Or, can local rather than systemic fashion use pharmaceutical composition, such as, by by pharmaceutical composition direct injection To the tissue regions of patient.

Orally administered is that the exemplary of tyrosine kinase inhibitor is used.Should be understood that tyrosine kinase inhibitor and anti- Using without going through identical path of WT-1 antibody, and need not perform simultaneously.

WT-1 (or anti-WT-1) antibody is by different for the properties of the antigen combined at it.Specificity is resisted by HLA Former type decided.Such as, HLA-A*0201 expresses in the white people of all 39-46%, therefore, WT-1 peptide is had specificity Antibody be combined with HLA-A2 and represent antibody appropriately selected used in white race crowd.For presenting on cancer cell surfaces WT-1 peptide there is specific anti-WT-1 antibody be combined with HLA-A24 that may to be particularly suited for wherein HLA-A24 target special The New World locals expressed and asian population.Therefore, the selection of WT-1 antibody, can depend on experimenter's extremely to be administered HLA type.

In other embodiments, anti-WT-1/HLA antibody can comprise one or more Framework Region amino acid and replace, institute State replace design for improving protein stability, antibodies, expression or introducing the site puted together for therapeutic agent. Then use these scFv to produce recombined human monoclonal Igs according to method known to those skilled in the art.

The method also including reducing Leukemia Cell Proliferation, described method includes the WT-1 making leukaemia with the present invention Antibody contacts.In a related aspect, the antibody of the present invention may be used for leukemic prevention or treatment.Executing of therapeutic antibodies With being known in the art.

The pharmaceutical composition for the treatment of and method

WT-1 antibody can be enough to prevent, suppress or reduce the amount of the progress of tumor or pathological condition and be applied to suffer from swollen The patient of tumor or WT-1-related pathologies condition of illness is so that treatment processes.Progress includes, the growth of such as tumor or pathological condition, invades Attack, shift and/or recur.The effective dose order of severity and the patient's self immune system that will depend upon which disease for this purposes General status.State along with morbid state and patient is also changed by dosage regimen, and generally will be at single-bolus high-dose Or in the range of injection is repeatedly to use (such as, every 4-6 hour) every day continuously, or as by the doctor in charge and status of patient indication Show.

By the WT-1 antibody of the present invention medicable medical treatment condition of illness qualification be those skilled in the art ability and In the ken.Such as, the individual human suffering from significantly leukemia or the clinically significant symptom of easy development clinically is suitable to execute With this WT-1 antibody.The skilled clinician of art technology such as, by the use of clinical trial, physical examination and medical treatment/ Family history can readily determine that, whether individual be the candidate of this treatment.

Feature is the limiting examples of the pathological condition that WT-1 expresses, and drenches including chronic myelocytic leukemia, acute one-tenth Bar cell leukemia (ALL), acute myelogenous/myelomatosis (AML) and myelodysplastic syndrome (MDS).Additionally, Solid tumor, it is however generally that and specifically, with mesothelioma, ovarian cancer, human primary gastrointestinal cancers, breast carcinoma, carcinoma of prostate and spongioblast The tumor that tumor is relevant is adapted in use to WT-1 Antybody therapy.

Any suitable method or approach can be used to use the WT-1 antibody of the present invention, and optionally, use altogether Antitumor agent and/or the antagonist of other receptor.Route of administration includes, such as, and oral, intravenous, intraperitoneal, subcutaneously or intramuscularly Inside use.But, it should emphasize, the invention is not restricted to any specific application process or approach.

It is understood that the WT-1 antibody of the present invention will be used in the form of compositions, described compositions also comprises medicine Acceptable carrier on.The pharmaceutically acceptable carrier being suitable for comprises such as, water, saline, phosphate buffered saline (PBS), dextrorotation One or more in sugar, glycerol, ethanol etc. with and combinations thereof.Pharmaceutically acceptable carrier can also comprise and strengthens knot on a small quantity The storage period of hop protein matter or the auxiliary substance of effectiveness, such as wetting agent or emulsifying agent, preservative or buffer agent.The group of injection Compound is permissible, prepares as known in the art, in order to provide the quick, lasting of active component after being applied to mammal Or the release postponed.

The other side of the present invention includes but not limited to, uses antibody and their nucleic acid of coding for treatment and WT-1 phase The disease closed, for diagnosis and prognosis application and with the research tool of detection of WT-1 in acting on cell and organizing.Comprise Disclosed antibody and the pharmaceutical composition of nucleic acid are covered by the present invention.The nucleic acid comprising the present invention for passing through vector immunity The carrier treated in antibody base of therapy, it is also possible to consider for the present invention.Carrier comprises to be enable the antibody express and secretes Expression vector, and the carrier of the cell surface expression for antigen-binding proteins (such as Chimeric antigen receptor).

Include but not limited in view of describing desired embodiment before, the embodiment below numbered:

1. a pharmaceutical composition, it comprises tyrosine kinase inhibitor (TKI)；And

(A) comprising heavy chain (HC) variable region and the antibody of light chain (LC) variable region, described heavy chain (HC) variable region comprises HC- CDR1, HC-CDR2 and HC-CDR3, and described light chain (LC) variable region comprises LC-CDR1, LC-CDR2 and LC-CDR3, described Heavy chain (HC) variable region and light chain (LC) variable region comprise the aminoacid sequence shown in table 1-14 and Fig. 7-10；Or

(B) V is comprised_HAnd V_LAntibody, described V_HAnd V_LComprise the first aminoacid sequence from table 1-12 and the second amino Acid sequence；Or

(C) comprising the antibody of scFv, described scFv comprises the aminoacid sequence from table 1-12.

2. the pharmaceutical composition as described in embodiment 1, wherein said tyrosine kinase inhibitor is selected from consisting of Group: imatinib, Dasatinib, AMN107, SKI-606, Ponatinib, Ba Fei for Buddhist nun, erlotinib, gefitinib, Lapatinib, Sorafenib and Sutent.

3. the pharmaceutical composition as described in embodiment 1, wherein said tyrosine kinase inhibitor is imatinib, Pu Na For Buddhist nun or Dasatinib or its pharmaceutically acceptable salt.

4. the pharmaceutical composition as described in embodiment 1, the pharmaceutically acceptable salt of wherein said imatinib is first Sulfonic acid imatinib.

5. the pharmaceutical composition as described in embodiment 1, the pharmaceutically acceptable salt of wherein said Ponatinib is salt Acid Ponatinib.

6. the method being used for treating or suppress the propagation of WT-1 positive cancer, described method includes being subject to in need The tyrosine kinase inhibitor of examination person's administering therapeutic effective dose and the anti-WT-1 antibody of therapeutically effective amount or its antigen binding fragment Section.

7. the method as described in embodiment 6, wherein said WT-1 positive cancer is selected from selected from the group consisted of: slow Property myelomatosis (CML), acute myelogenous/myelomatosis (AML), acute lymphoblastic leukemia (ALL) and Myelodysplastic syndrome (MDS), gastrointestinal stromal tumor, ovarian cancer, carcinoma of prostate, soft tissue sarcoma and glioblastoma.

8. the method as described in embodiment 6, wherein said tyrosine kinase inhibitor is selected from the group consisted of: she Imatinib, Dasatinib, AMN107, SKI-606, Ponatinib, Ba Fei replace Buddhist nun, erlotinib, gefitinib, draw handkerchief to replace Buddhist nun, Sorafenib and Sutent.

9. the method as described in embodiment 6, wherein said tyrosine kinase inhibitor be imatinib, Ponatinib or Dasatinib or its pharmaceutically acceptable salt.

10. the method as described in embodiment 6, the pharmaceutically acceptable salt of wherein said imatinib be methanesulfonic acid she Imatinib.

11. methods as described in embodiment 6, the pharmaceutically acceptable salt of wherein said Ponatinib is hydrochloric acid Pu Na For Buddhist nun.

12. methods as described in embodiment 6, wherein said anti-WT-1 antibody is selected from the group consisted of:

(A) comprising heavy chain (HC) variable region and the antibody of light chain (LC) variable region, described heavy chain (HC) variable region comprises HC- CDR1, HC-CDR2 and HC-CDR3, and light chain (LC) variable region comprises LC-CDR1, LC-CDR2 and LC-CDR3, described heavy chain (HC) variable region and light chain (LC) variable region comprise the aminoacid sequence shown in table 1-14 and Fig. 7-10；Or

(B) V is comprised_HAnd V_LAntibody, described V_HAnd V_LComprise the first aminoacid sequence from table 1-12 and the second amino Acid sequence；Or

(C) comprising the antibody of scFv, described scFv comprises the aminoacid sequence from table 1-12.

13. methods as described in embodiment 6, wherein said anti-WT-1 antibody comprises people's variable domain framework region.

14. methods as described in embodiment 6, wherein said anti-WT-1 antibody is fully human antibodies.

15. methods as described in embodiment 6, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, limit with HLA Property mode processed specific binding WT-1 peptide.

16. methods as described in embodiment 6, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, with 1 × 10^-8The K of M or less_DIt is bound to WT-1/HLA.

17. methods as described in embodiment 6, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, with about 1 × 10^-11M to about 1 × 10^-8The K of M_DIt is bound to WT-1/HLA.

18. methods as described in embodiment 6, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, induce pin Antibody dependent cellular cytotoxicity (ADCC) to WT-1-positive cell.

19. methods as described in embodiment 6, wherein said anti-WT-1 antibody, or the suppression of its antigen-binding portion thereof is internal The growth of WT-1 positive cell.

20. methods as described in embodiment 6, the Fab of wherein said antibody is Fab, Fab', F (ab')₂, Fv or scFv (scFv).

Now by relative to the method being more fully described the present invention in representative embodiment.

Embodiment 1

Material and method

Cell sample, cell line and antibody.In signature about Si Long-Caitlin Cancer center institutional review board Knowing the inside story of the agreement that (Sloan-Kettering Cancer Center Institutional Review Board) ratifies is same After meaning, obtain obtaining peripheral blood lymphocytes from HLA-type of health donor and patient by Ficoll density gradient centrifugation (PBMC).All cells is by commemorating Si Long-Caitlin Cancer center (Memorial Sloan-Kettering Cancer Center) cellular immunology teaching and research room carries out HLA typing.Leukemia Cell Lines BV173 (WT-1+, A0201+) by Doctor H.J.Stauss (University College London, London, United Kingdom) presents.37 DEG C/ 5%CO₂Under, cell line is being supplemented with 5%FCS, penicillin, streptomycin, 2mmol/L glutamine and 2 mercapto ethanol RPMI 1640 cultivates.

Animal.The male NOD.Cg-Prkdc scid IL2rgtm1Wjl/SzJ mice that six to eight weeks are big, is referred to as NOD/ SCID γ (NSG), is purchased from Jackson Laboratory (Bar Harbor, ME) or obtains from MSKCC animal breeding plant.

The transduction of luciferase/GFP positive cell and selection.The slow virus using the plasmid containing coding luc/GFP carries Body, engineering design BV173 cell is to express high-caliber GFP-Luciferase fusion albumen (39).Use single cell clone, logical Overflow-type Cytometric Analysis only selects to illustrate the cell that high level GFP expresses, and keeps and for zooscopy.

Embodiment 2

Antibody dependent cellular cytotoxicity (ADCC)

ADCC is considered as the one in the main effects mechanism for the treatment of people mAb.Therefore, the assessment of curative effect, start from surveying The metering pin testing in vitro to the ADCC of BV173 cell line, described BV173 cell line derives from the CML of initiation potential phase.Fresh BV173 cell is used for ADCC target cell.By sub in different effect at 750ng/ml to WT-1 antibody or its isotype controls human IgG1: Hatch together with target cell and fresh PBMC under target (E:T) ratio, keep 6 hours.Interpolation concentration is she of 0,1,5 and 10 μMs Imatinib.Gather in the crops supernatant and measure cytotoxicity by standard Cr51 release mensuration.

In the presence of human PBMC, the antibody-mediated dose dependent PBMC for the RMF epi-position naturally presented of WT-1 ADCC, described RMF epi-position is presented naturally by the HLA-A0201 molecule on tumor cell (Leukemia Cell Lines BV173).Weight , in the presence of the imatinib of various dose, WT-1 antibody can mediate ADCC.Use from multiple healthy donors PBMC action effect cell, unanimously observes when WT-1 antibody is 750ng/ml and kills.These results indicate that imatinib is not Affecting the ability of the antibody-mediated specific ADCC for cell of WT-1, described cell expresses RMF and HLA-A0201 the most naturally Complex (Fig. 1).

Embodiment 3

Injection is used to have HLA-A0201⁺The NSG mice of Leukemia Cell Lines BV173 assesses the internal treatment of ESKM Yu TKI Effect.Scheme for the imatinib being combined with ESKM and Dasatinib therapy include every mice by tail vein injection 3 × 10⁶Individual cell, injects fluorescein imaging in latter 6 days to evaluate tumour transplatation, and immediately begins to treatment after imaging in the 6th day.Often Use fluorescein imaging to monitor tumor growth in week.Every day peritoneal injection TKI (imatinib of 50mg/kg and 20-40mg/ The Dasatinib of kg.) antibody described in intravenous injection twice a week.

Embodiment 4

Imatinib adds the anti-WT-1/HLA antibody (ESKM) curative effect in human leukemia xenotransplantation NSG model.Will 3000000 BV173 human leukemia cells pass through tail vein injection to NSG mice.At the 6th day, in all mices to be treated Tumour transplatation is confirmed by LUC Photinus pyralis LUC Photinus pyralis FL imaging；Then mice is randomly divided into different treatment group (A, B, C and D).After imaging the 6th day immediately, use 100 μ g anti-WT-1 antibody ESKM start to control twice weekly by intraperitoneal (IP) injection Treat.Every day uses 50mg/kg imatinib also by IP injection.Treatment continues 5 weeks (ESKM of every mice 10 dosage and 34 doses The imatinib of amount).Group A: do not treat；Group B: only treatment with imatinib；Group C: only ESK treatment；Group D: her horse once a day For Buddhist nun and the combination of semiweekly ESK.Assess tumor growth by luminescence imaging weekly, and assess clinical activity every day.

After treatment 5 weeks, animal imaging by fluorescein fluorescence imaging, and use LivingSoftware quantification Described fluorescence.This allows quantitative to mouse tumor load.Result figure 3 illustrates.With do not receive imatinib or anti-WT-1 The control animal of antibody is compared, and the animal only receiving imatinib (50mg/kg every day) has the tumor load of reduction.Accept every Week, twice 100 μ g anti-WT-1 antibody animal of continuing 5 weeks bright compared control mice and treatment with imatinib mice is aobvious improves very Many.Minimizing discovery in the animal accepting anti-WT-1 antibody and imatinib combination (group D) that tumor cell is maximum.These move Thing also shows the tumor growth of reduction, and before one week, the previous day of they imagings is more apparent.(Fig. 2)

Embodiment 5

Dasatinib adds the anti-WT-1/HLA antibody (ESKM) curative effect in human leukemia xenotransplantation NSG model.Will 3000000 BV173 human leukemia cells pass through tail vein injection to NSG mice.At the 6th day, in all mices to be treated Tumour transplatation is confirmed by LUC Photinus pyralis LUC Photinus pyralis FL imaging；Then mice is randomly divided into 5 different treatment groups (A, B, C, D And E).After imaging the 6th day immediately, use 100 μ g anti-WT-1 antibody ESKM start twice weekly by intraperitoneal (IP) injection Treatment.Every day uses 40mg/kg Dasatinib also by IP injection.Because Dasatinib is the most soluble, it is molten Solution is used in 50 μ L DMSO.Group A: do not treat；Group B: only DMSO (vehicle Control)；Group C: only Dasatinib treatment；Group D: Only ESK treats；Group E: Dasatinib once a day and the combination of semiweekly ESK.After treating 7 days, it is noted that with reaching sand Mice for Buddhist nun's treatment seems to be out of shape, and body weight substantially alleviates.Dosage reduces to 20mg/kg.Mice is still in a bad state of health, 1 death, and treat owing to toxicity stopped Dasatinib at the 11st day.ESK antibody persistently uses full 4 weeks treatment cycle.Often Week continues assessment tumor growth by luminescence imaging.

After treatment 4 weeks, animal imaging by fluorescein fluorescence imaging, and use LivingAmount of software Change described fluorescence, quantify mouse tumor load.Result figure 6 illustrates.Originally there is tumor in the animal only accepting Dasatinib Remove, but be reoccurred through the treatment of 4 weeks.Accept the animal phase comparison that 100 μ g anti-WT-1 antibody continues 4 weeks twice a week Being obviously improved much according to mice, although compared with the mice only using Dasatinib, they have the tumor load of increase.Accept In the mice of Dasatinib and ESK combination, a mouse intracranial tumor recurrence, and other three mices keep without tumor.5th Mice dies from Dasatinib toxicity on the 8th day treatment.

Embodiment 6

BV173 is HLA-A0201+, is the people's Ph+ALL cell line expressing WT1, and by luciferase labelling.By with Resistance T315I Bcr-Abl plasmid transduction BV173 carrys out through engineering approaches tyrosine kinase inhibitor (TKI) resistance BV173-R cell line. By chromium release assay, utilize human PBMC's effector to assess antibody dependent cellular cytotoxicity (ADCC).Use bioluminescence becomes As (BLI) evaluates tumor growth in vivo in NOD/SCID γ (NSG) mice.RT-PCR is used to have negative latter stage with assessment treatment Minimum residual disease in the mice of BLI signal.Imatinib, Dasatinib and Pu Na is used to replace reaching maximum tolerated dose Buddhist nun, every day, IP was administered once.IP uses the ESKM of 100 μ g twice a week.

The external mediation of ESKM is for the ADCC of BV173 and BV173-R cell line.At the human leukemia xenogenesis that BV173 transplants In graft model, ESKM is more more effective than imatinib, and the median tumor growth with 78% (to 52%) reduces.Imatinib Combination with ESKM therapy causes leukemia to increase reduction by 94%.High dose Dasatinib (40mg/kg every day) more has than ESKM Effect, but owing to Dasatinib toxicity causes recurrence to stop treatment.ESKM therapy causes 75% mice with the combination of Dasatinib Curing, this is confirmed by bone marrow RT-PCR after stopping treatment three weeks.For using Dasatinib cytoreductive then to use ESKM The mice of after treatment, it was observed that postpone recurrence, but do not cure.For internal T315I BV173-R leukemia, ESKM height It is better than imatinib and Dasatinib.Do not have for resistance T315I leukemia, ESKM and first or second filial generation TKI combination treatment Realize curing.

The Ponatinib of 10mg/kg has than independent ESKM for the more preferable effect of BV173-R, but uses ESKM and Pu Na Mice for Buddhist nun's combined therapy has higher tumor minimizing (Figure 13).Conclusion be ESKM be for sensitive and T315I Ph+ ALL effectively treats antibody.Compared with imatinib and Dasatinib, by the ESKM treatment suppression white blood of resistance T315I Ph+ Sick growth is more effective, and is better than Ponatinib with ESKM combination treatment.

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Claims (21)

1. a pharmaceutical composition, it comprises tyrosine kinase inhibitor (TKI)；And

(A) comprising heavy chain (HC) variable region and the antibody of light chain (LC) variable region, described heavy chain (HC) variable region comprises HC- CDR1, HC-CDR2 and HC-CDR3, and described light chain (LC) variable region comprises LC-CDR1, LC-CDR2 and LC-CDR3, described Heavy chain (HC) variable region and described light chain (LC) variable region comprise the aminoacid sequence shown in table 1-14 and Fig. 7-10；Or

(B) V is comprised_HAnd V_LAntibody, described V_HAnd V_LComprise the first aminoacid sequence from table 1-12 and the second aminoacid sequence Row；Or

(C) comprising the antibody of scFv, described scFv comprises the aminoacid sequence from table 1-12.

2. pharmaceutical composition as claimed in claim 1, wherein said tyrosine kinase inhibitor is selected from the group consisted of: Imatinib, Dasatinib, AMN107, SKI-606, Ponatinib, Ba Fei replace Buddhist nun, erlotinib, gefitinib, La Pa For Buddhist nun, Sorafenib and Sutent.

3. pharmaceutical composition as claimed in claim 1, wherein said tyrosine kinase inhibitor is imatinib, Ponatinib Or Dasatinib or its pharmaceutically acceptable salt.

4. pharmaceutical composition as claimed in claim 1, the pharmaceutically acceptable salt of wherein said imatinib is methanesulfonic acid Imatinib.

5. pharmaceutical composition as claimed in claim 1, the pharmaceutically acceptable salt of wherein said Ponatinib is that hydrochloric acid is general Receive for Buddhist nun.

6. tyrosine kinase inhibitor and one anti-WT-1 antibody or its Fab, it is used for treating or suppressing The propagation of WT-1 positive cancer.

7. the method being used for treating or suppress the propagation of WT-1 positive cancer, described method includes to experimenter in need The tyrosine kinase inhibitor of administering therapeutic effective dose and the anti-WT-1 antibody of therapeutically effective amount or its Fab.

Purposes the most as claimed in claims 6 or 7 or method, wherein said WT-1 positive cancer is selected from the group consisted of: Chronic lymphocytic leukemia (CML), acute myeloid leukaemia (AML), acute lymphoblastic leukemia (ALL) and bone marrow Hypertrophy exception syndrome (MDS), gastrointestinal stromal tumor, ovarian cancer, carcinoma of prostate, soft tissue sarcoma and glioblastoma.

Purposes the most as claimed in claims 6 or 7 or method, wherein said tyrosine kinase inhibitor is selected from consisting of Group: imatinib, Dasatinib, AMN107, SKI-606, Ponatinib, Ba Fei replace Buddhist nun, erlotinib, gefitinib, draw Handkerchief is for Buddhist nun, Sorafenib and Sutent.

Purposes the most as claimed in claims 6 or 7 or method, wherein said tyrosine kinase inhibitor is imatinib, Pu Na For Buddhist nun or Dasatinib or its pharmaceutically acceptable salt.

11. purposes as claimed in claims 6 or 7 or method, the pharmaceutically acceptable salt of wherein said imatinib is first Sulfonic acid imatinib.

12. purposes as claimed in claims 6 or 7 or method, the pharmaceutically acceptable salt of wherein said Ponatinib is salt Acid Ponatinib.

13. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody is selected from the group consisted of:

(A) comprising heavy chain (HC) variable region and the antibody of light chain (LC) variable region, described heavy chain (HC) variable region comprises HC- CDR1, HC-CDR2 and HC-CDR3, and described light chain (LC) variable region comprises LC-CDR1, LC-CDR2 and LC-CDR3, described Heavy chain (HC) variable region and light chain (LC) variable region comprise the aminoacid sequence shown in table 1-14 and Fig. 7-10；Or

(B) V is comprised_HAnd V_LAntibody, described V_HAnd V_LComprise the first aminoacid sequence from table 1-12 and the second aminoacid sequence Row；Or

(C) comprising the antibody of scFv, described scFv comprises the aminoacid sequence from table 1-12.

14. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody comprises people's variable domain framework region.

15. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody is fully human antibodies.

16. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, with HLA restrictive one specific binding WT-1 peptide.

17. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, with 1×10^-8The K of M or less_DIn conjunction with WT-1/HLA.

18. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, with About 1 × 10^-11M to about 1 × 10^-8The K of M_DIn conjunction with WT-1/HLA.

19. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, lure The guide pin antibody dependent cellular cytotoxicity (ADCC) to WT-1-positive cell.

20. purposes as claimed in claims 6 or 7 or method, wherein said anti-WT-1 antibody, or its antigen-binding portion thereof, press down Make the growth of internal WT-1 positive cell.

21. purposes as claimed in claims 6 or 7 or method, the Fab of wherein said antibody is Fab, Fab', F (ab')₂, Fv or scFv (scFv).